Process for enzymatic hydrolysis of lignocellulosic material and fermentation of sugars

ABSTRACT

The invention relates to a process for the preparation of a fermentation product from ligno-cellulosic material, comprising the following steps:
     a) optionally pre-treatment of the ligno-cellulosic material;   b) optionally washing of the optionally pre-treated ligno-cellulosic material;   c) enzymatic hydrolysis of the optionally washed and/or optionally pre-treated ligno-cellulosic material using an enzyme composition comprising at least two cellulase and whereby the enzyme composition at least comprises GH61;   d) whereby less than 7.5 mg enzyme composition/g glucan (on dry matter and enzyme as protein) or less than 3.0 mg enzyme composition/g feedstock (on dry matter and enzyme as protein) is used; and   e) fermentation of the hydrolysed ligno-cellulosic material to produce a fermentation product; and   f) optionally recovery of a fermentation product;
 
wherein before and/or during the enzymatic hydrolysis oxygen is added to the ligno-cellulosic material.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.16/152,197, filed 4 Oct. 2018, which is a Continuation of U.S.application Ser. No. 15/956,841, filed 19 Apr. 2018, now U.S. Pat. No.10,131,923, issued 20 Nov. 2018, which is a Continuation of U.S.application Ser. No. 14/440,662, filed on 5 May 2015, now U.S. Pat. No.9,982,280, issued 29 May 2018, which is a § 371 National StageApplication of PCT/EP2013/073255, filed 7 Nov. 2013, which claimspriority to EP 12191957.5, filed 9 Nov. 2012, EP 13174656.2, filed 2Jul. 2013, EP 13176083.7, filed 11 Jul. 2013, EP 13176500.0, filed 15Jul. 2013, EP 13184702.2, filed 17 Sep. 2013 and EP 13184701.4, filed 17Sep. 2013. Each of these applications is incorporated by reference inits entirety.

The invention relates to a process for the enzymatic hydrolysis oflignocellulosic material and fermentation of sugars.

DESCRIPTION OF RELATED ART

Ligno-cellulosic plant material, herein also called feedstock, is arenewable source of energy in the form of sugars that can be convertedinto valuable products e.g. sugars or bio-fuel, such as bio-ethanol.During this process, (ligno or hemi)-cellulose present in the feedstock,such as wheat straw, corn stover, rice hulls, etc., is converted intoreducing sugars by (hemi)-cellulolytic enzymes, which then areoptionally converted into valuable products such as ethanol bymicroorganisms like yeast, bacteria and fungi.

Since the (hemi)-cellulose is crystalline and entrapped in a network oflignin the conversion into reducing sugars is in general slow andincomplete. Typically, enzymatic hydrolysis of untreated feedstockyields sugars <20% of theoretical quantity. By applying a chemical andthermo-physical pre-treatment, the (hemi)-cellulose is more accessiblefor the (hemi)-cellulolytic enzymes, and thus conversions go faster andat higher yields.

A typical ethanol yield from glucose, derived from pre-treated cornstover, is 40 gallons of ethanol per 1000 kg of dry corn stover (Badger,P, Ethanol from cellulose: a general review, Trends in new crops and newuses, 2002, J. Janick and A. Whipkey (eds.) ASHS Press, Alexandria, Va.)or 0.3 g ethanol per g feedstock. The maximum yield of ethanol oncellulose base is approximately 90%.

Cellulolytic enzymes—most of them are produced by species likeTrichoderma, Humicola and Aspergillus—are commercially used to convertpre-treated feedstock into a mash containing insoluble (hemi)cellulose,reducing sugars made thereof, and lignin. Thermostable cellulolyticenzymes derived from Rasamsonia, have been used for degradingligno-cellulosic feedstock and these enzymes are known for theirthermostability, see WO2007091231. The produced mash is used in afermentation during which the reducing sugars are converted into yeastbiomass (cells), carbon dioxide and ethanol. The ethanol produced inthis way is called bio-ethanol.

The common production of sugars from pre-treated ligno-cellulosicfeedstock, the hydrolysis also called liquefaction, pre-saccharificationor saccharification, typically takes place during a process lasting 6 to168 hours (Kumar, S., Chem. Eng. Technol. 32 (2009) 517-526) underelevated temperatures of 45 to 50° C. and non-sterile conditions. Duringthis hydrolysis, the cellulose present is partly (typically 30 to 95%,dependable on enzyme activity and hydrolysis conditions) converted intoreducing sugars. In case of inhibition of enzymes by compounds presentin the pre-treated feedstock and by released sugars; and to minimizethermal inactivation, this period of elevated temperature is minimizedas much as possible.

The fermentation following the hydrolysis takes place in a separatepreferably anaerobic process step, either in the same or in a differentvessel, in which temperature is adjusted to 30 to 33° C. (mesophilicprocess) to accommodate growth and ethanol production by microbialbiomass, commonly yeasts. During this fermentation process, theremaining (hemi) cellulosic material is converted into reducing sugarsby the enzymes already present from the hydrolysis step, while microbialbiomass and ethanol are produced. The fermentation is finished once(hemi) cellulosic material is converted into fermentable sugars and allfermentable sugars are converted into ethanol, carbon dioxide andmicrobial cells. This may take up to 6 days. In general the overallprocess time of hydrolysis and fermentation may amount up to 13 days.

The so obtained fermented mash consists of non-fermentable sugars,non-hydrolysable (hemi) cellulosic material, lignin, microbial cells(most common yeast cells), water, ethanol, dissolved carbon dioxide.During the successive steps, ethanol is distilled from the mash andfurther purified. The remaining solid suspension is dried and used as,for instance, burning fuel, fertilizer or cattle feed.

WO2010080407 suggests treating cellulosic material with a cellulasecomposition under anaerobic conditions. Removal or exclusion of reactiveoxygen species may improve the performance of cellulose-hydrolyzingenzyme systems. Hydrolysis of cellulosic material, e.g., lignocellulose,by an enzyme composition can be reduced by oxidative damage tocomponents of the enzyme composition and/or oxidation of the cellulosicmaterial by, for example, molecular oxygen.

WO2009046538 discloses a method for treating lignocellulosic feedstockplant materials to release fermentable sugars using an enzymatichydrolysis process for treating the materials performed under vacuum andproducing a sugar rich process stream comprising reduced amounts ofvolatile sugar/fermentation inhibiting compounds such as furfural andacetic acid. Apart from removing volatile inhibitory compounds, othercompounds and/or molecules that are also removed include nitrogen,oxygen, argon and carbon dioxide.

With each batch of feedstock, enzymes are added to maximize the yieldand rate of fermentable sugars released from the pre-treatedligno-cellulosic feedstock during the given process time. In general,costs for enzymes production, feedstock to ethanol yields andinvestments are major cost factors in the overall production costs(Kumar, S. Chem. Eng. Technol. 32 (2009) 517-526). Thus far, cost ofenzyme usage reduction is achieved by applying enzyme products from asingle or from multiple microbial sources (WO 2008/008793) with broaderand/or higher (specific) hydrolytic activity which use aims at a lowerenzyme need, faster conversion rates and/or a higher conversion yields,and thus at overall lower bio-ethanol production costs. This requireslarge investments in research and development of these enzyme products.In case of an enzyme product composed of enzymes from multiple microbialsources, large capital investments are needed for production of eachsingle enzyme compound.

It is therefore desirable to improve the above process involvinghydrolysis and fermentation.

SUMMARY

An object of the invention is therefore to provide a process in whichthe hydrolysis step is conducted at improved conditions. Another objectof the invention is to provide a process involving hydrolysis having areduced process time. Further object of the invention is to provide aprocess, wherein the dosage of enzyme may be reduced and at the sametime output of useful hydrolysis product is maintained at the same levelor even increased. Another object is to provide a process involvinghydrolysis, wherein the process conditions of the hydrolysis areoptimized. A still further object of the invention is to provide aprocess involving hydrolysis, wherein the output of useful hydrolysisproduct is increased using the same enzyme dosage. One or more of theseobjects are attained according to the invention.

The present invention provides a process for the preparation of a sugarproduct from ligno-cellulosic material, comprising the following steps:

-   -   a) optionally pre-treatment of the ligno-cellulosic material;    -   b) optionally washing of the optionally pre-treated        ligno-cellulosic material;    -   c) enzymatic hydrolysis of the optionally washed and/or        optionally pre-treated ligno-cellulosic material using an enzyme        composition comprising at least two cellulases and whereby the        enzyme composition at least comprises GH61;    -   d) whereby less than 7.5 mg enzyme composition/g glucan (on dry        matter and enzyme as protein) or less than 3.0 mg enzyme        composition/g feedstock (on dry matter and enzyme as protein) is        used; and    -   e) optionally recovery of a sugar product;

wherein after the pre-treatment and before and/or during the enzymatichydrolysis oxygen is added to the ligno-cellulosic material.

Preferably during the enzymatic hydrolysis step c) oxygen is added tothe ligno-cellulosic material.

According to an embodiment of the invention during part of the time ofthe enzymatic hydrolysis, oxygen is added to the ligno-cellulosicmaterial and during part of the time of the enzymatic hydrolysis lessoxygen is added to the ligno-cellulosic material compared to the otherpart of the time of the enzymatic hydrolysis, preferably no oxygen isadded to the ligno-cellulosic material.

Furthermore the present invention provides a process for the preparationof a fermentation product from ligno-cellulosic material, comprising thefollowing steps:

-   -   a) optionally pre-treatment of the ligno-cellulosic material;    -   b) optionally washing of the optionally pre-treated        ligno-cellulosic material;    -   c) enzymatic hydrolysis of the optionally washed and/or        optionally pre-treated ligno-cellulosic material using an enzyme        composition comprising at least two cellulases and whereby the        enzyme composition at least comprises GH61;    -   d) fermentation of the hydrolysed ligno-cellulosic material to        produce a fermentation product;    -   e) whereby less than 7.5 mg enzyme composition/g glucan (on dry        matter and enzyme as protein) or less than 3.0 mg enzyme        composition/g feedstock (on dry matter and enzyme as protein) is        used; and    -   f) optionally recovery of a fermentation product;        wherein after the pre-treatment and before and/or during the        enzymatic hydrolysis oxygen is added to the ligno-cellulosic        material.

Preferably during the enzymatic hydrolysis step c) oxygen is added tothe ligno-cellulosic material.

According to an embodiment of the invention during part of the time ofthe enzymatic hydrolysis, oxygen is added to the ligno-cellulosicmaterial and during part of the time of the enzymatic hydrolysis lessoxygen is added to the ligno-cellulosic material compared to the otherpart of the time of the enzymatic hydrolysis, preferably no oxygen isadded to the ligno-cellulosic material.

According to a preferred embodiment of the invention the part of thetime wherein less or preferably no oxygen is added is 10 to 80%,preferably 20 to 80%, more preferably 30 to 80% and most preferably 40to 80% of the total enzymatic hydrolysis time.

According to another preferred embodiment of the invention the part ofthe time wherein more oxygen is added is 2 to 80%, preferably 4 to 60%,more preferably 8 to 50% and most preferably 10 to 50% of the totalenzymatic hydrolysis time, more preferably the part of the time whereinmore oxygen is added is

-   -   a) 12 to 50%, and preferably 20 to 40% when the oxygen is added        in the second half of time of the enzymatic hydrolysis;    -   b) 2 to 30%, preferably 4 to 25% and more preferably 5 to 20% of        the total enzymatic hydrolysis time when the oxygen is added in        the first half of time of the enzymatic hydrolysis; or    -   c) or a combination of a and b.

Advantageously the oxygen concentration in the liquid phase of thehydrolysis during the part of the time wherein oxygen is added is atleast 2 times, preferably at least 4 times, more preferably at least 10times the oxygen concentration in the liquid phase during the part ofthe time wherein less or no oxygen is added.

According to a further preferred embodiment of the invention, in thepart of the time when the oxygen is added, the oxygen concentration inthe liquid phase, wherein the ligno-cellulosic material is presentduring the enzymatic hydrolysis, is at least 0.001 mol/m³, preferably atleast 0.002 mol/m³ and most preferably at least 0.003 mol/m³ and evenmore preferably more than 0.01 mol/m³, for example more than 0.02 mol/m³or 0.03 mol/m³. In reactors of less than 1 m³ DO values of below 0.01mol/m³ or 0.02 mol/m³ will be obtained by slow stirring. Vigorous mixingor stirring at such scale introduces part of the gas phase of theheadspace into the reaction liquid. For example the mixing or stirringmay create a whirlpool that draws oxygen into the liquid. In generalflushing the headspace with air in combination with (vigorous) mixing orstirring will introduce sufficient oxygen into the cellulosic materialin the hydrolysis reactor for reactors up to a size of 100 liter to 1m³. At larger scale, for example in a reactor of 50 m³ or more, forexample 100 m³, so much energy is needed for vigorous stirring that fromeconomic point of view this will not be applied in a commerciallyoperating process. In general in large reactors, stirring or mixingwithout introducing air or oxygen will result in DO values of less than0.01 mol/m³.

To still another preferred embodiment of the invention during the oxygenaddition (in the part of the time when the oxygen is added), the oxygenconcentration in the liquid phase, wherein the ligno-cellulosic materialis present during the enzymatic hydrolysis, is preferably at most 80% ofthe saturation concentration of oxygen under the hydrolysis reactionconditions, more preferably at most 0.12 mol/m³, still more preferablyat most 0.09 mol/m³, even more preferably at most 0.06 mol/m³, evenstill more preferably at most 0.045 mol/m³ and most preferably at most0.03 mol/m³. Temperature and pressure will influence the DO. Thepreferred and exemplary mol/m³ values given above relate to normalatmospheric pressure and a temperature of about 62° C. The skilledperson in the art will appreciate favourable DO values on basis of thepresent teachings.

According to another preferred embodiment of the invention the reactorfor the enzymatic hydrolysis has a volume of 1 m³ or more. The enzymatichydrolysis time of the present process is preferably from 5 to 150hours. According to a further preferred aspect of the invention theenzyme composition is derived from a fungus, preferably a microorganismof the genus Rasamsonia or the enzyme composition comprises a fungalenzyme, preferably a Rasamsonia enzyme. According to a still furtherpreferred aspect of the invention the dry matter content in thehydrolysis step c) is 10 wt % or more, preferably 14 wt % or more andstill more preferably from 14 to 33% wt %. The enzymatic hydrolysispreferably takes place in a batch, fed batch and/or continuous culturereactor. Preferably the oxygen that is introduced in the present processis an oxygen-containing gas such as air. By less oxygen is added to oris present in to the ligno-cellulosic material during part of the timeof the enzymatic hydrolysis, is meant that at least 50% less, preferablyat least 70% less, most preferably at least 90% less of oxygen(expressed in mol oxygen/m³) is introduced, for example in bubble-formor is present than is added or is present during to the other part ofthe time of the enzymatic hydrolysis wherein less oxygen is added.

In a preferred embodiment the oxygen is added in the form of (gaseous)bubbles.

Surprisingly, according to the invention, by the addition of oxygen itis possible to attain many process advantages, including optimaltemperature conditions, reduced process time, reduced dosage of enzyme,re-use of enzymes, higher yields and other process optimizations,resulting in reduced costs.

In an embodiment the stable enzyme composition used retains activity for30 hours or more. According to a further embodiment the hydrolysis ispreferably conducted at a temperature of 45° C. or more, preferably at atemperature of 50° C. or more and more preferably at a temperature of55° C. or more. The process of the invention will be illustrated in moredetail below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: The effect of sparging nitrogen or air through a 10% aCSfeedstock before hydrolysis, on the total amount of glucose (g/l)released by the TEC-210 mix (1), 4E-GH61 mix (2) and 4E-EG mix (3).

FIG. 2: The glucose produced in Example 2, 1=Experiment 1: no aeration,2=Experiment 2: continuous aeration, 3=Experiment 3: aeration startingat 72 hours until the end

FIG. 3: The effect of time of aeration on glucose produced duringenzymatic hydrolysis, — —=no aeration, ●●●=aeration between hydrolysistime is 0 and 100 hours, - - - aeration between hydrolysis-time is 0 and7 hours and — — —=aeration between hydrolysis-time is 72 and 100 hours

FIG. 4: The effect of time of aeration on glucose produced duringenzymatic hydrolysis in experiment 1 (▪=aeration between hydrolysis-timeis 0 and 100 hours) and 2 (□=aeration between hydrolysis-time is 72 and100 hours)

FIG. 5: The effect of time of aeration on glucose produced duringenzymatic hydrolysis, —▪— aeration between hydrolysis-time is 72 and 100hours and —●— aeration between hydrolysis-time is 0 and 7 hours

FIG. 6: The effect of the dissolved oxygen concentration (DO) on glucanhydrolysis in pretreated lignocellulosic feedstock as function ofhydrolysis time for 2.5 mg/g of enzyme and DO=0.030 mol/m³ (—♦—) and 3.5mg/g of enzyme and DO=0.005 mol/m³ (—▪—).

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

Throughout the present specification and the accompanying claims, thewords “comprise” and “include” and variations such as “comprises”,“comprising”, “includes” and “including” are to be interpretedinclusively. That is, these words are intended to convey the possibleinclusion of other elements or integers not specifically recited, wherethe context allows. The articles “a” and “an” are used herein to referto one or to more than one (i.e. to one or at least one) of thegrammatical object of the article. By way of example, “an element” maymean one element or more than one element.

In the context of the present invention “improved”, “increased”,“reduced” is used to indicate that the present invention shows anadvantage compared to the same situation, process or process conditionsexcept that no extra oxygen is added. Within the context of the presentinvention “measured under the same conditions” or “analysed under thesame conditions” etc. means that the process of the invention and thesame process without (or with less) addition of oxygen are performedunder the same conditions (except the oxygen addition) and that theresults of the present process, if compared to the process without (orwith less) oxygen addition, are measured using the same conditions,preferably by using the same assay and/or methodology, more preferablywithin the same or parallel experiment. Conditions of the hydrolysis arean example of such conditions.

In prior art it is suggested to improve the hydrolysis of cellulolyticmaterial by using anaerobic (WO2010/080407) or vacuum (WO2009/046538)conditions during the enzymatic hydrolysis. In the processes of bothdocuments the oxygen level was decreased. It has been surprisingly foundthat the hydrolysis of the present invention shows results in animproved reaction product that gives higher amounts of (reduced) sugarproducts and/or desired fermentation products in the fermentationfollowing the hydrolysis as compared to a process wherein no oxygen isadded. In general an increase of the glucose conversion is observed of 5to 15 w/w %, or even up to 25 w/w %.

Oxygen can be added in several ways. For example oxygen can be added asoxygen gas, oxygen enriched gas such as oxygen enriched air or air(example of oxygen containing gas). Oxygen can be added continuously ordis-continuously. By oxygen “is added” is meant that oxygen is added tothe liquid phase (comprising the ligno-cellulosic material) in thehydrolysis reactor and not that oxygen is present in the headspace inthe reactor above the liquid phase (in combination with slow or nostirring) whereby the oxygen has to diffuse from the headspace to theliquid phase. So preferably the oxygen is added as bubbles, mostpreferably as small bubbles.

In case the enzyme may be damaged by the presence or addition of oxygen,milder oxygen supply may be used. In that case a balance can be foundbetween the improved glucose production and the enzyme performance. Theaddition of the oxygen to the cellulolytic material can be done duringthe enzymatic hydrolysis. In case oxygen is added in gaseous form,oxygen-containing gas can be introduced, for example blown, into theliquid hydrolysis reactor contents of cellulolytic material. In anotherembodiment of invention the oxygen-containing gas is introduced into theliquid cellulolytic material stream that will enter the hydrolysisreactor. In still another embodiment of the invention the oxygencontaining gas is introduced together with the cellulolytic materialthat enters the hydrolysis reactor or with part of the liquid reactorcontents that passes an external loop of the reactor. In most cases theaddition of oxygen before entering the hydrolysis reactor is notsufficient enough and oxygen addition may be done during the hydrolysisas well. In another embodiment of the invention the gaseous phasepresent in the upper part of the reactor (head space) is continuously ordis-continuously refreshed with the oxygen-containing gas. In the lattercase (vigorous) mixing or stirring is needed to get the oxygen asbubbles and/or by diffusion into the liquid reactor contents preferablyin combination with over-pressure in the reactor. In general flushingthe headspace with air in combination with (vigorous) mixing or stirringmay introduce sufficient oxygen into the cellulosic material in thehydrolysis reactor for reactors up to a size of 100 liter to 1 m³. Atlarger scale, for example in a reactor of 50 m³ or more, for example 100m³, so much energy is needed for vigorous stirring that from economicpoint of view this will not be applied in a commercially operatingprocess.

According to the present invention the oxygen may be added before thehydrolysis step, during part of the hydrolysis step, during the wholehydrolysis step or a combination of before or during the hydrolysisstep. Advantageously the oxygen is added during part of the hydrolysisstep. The addition of oxygen during only part of the hydrolysis may bedone for example in case of oxidation damage of the enzyme(s) occurs. Incase the oxygen present in the hydrolysis reactor contents or the sugarproduct or hydrolysate formed in the hydrolysis step might influence ordisturb in the subsequent fermentation step, oxygen addition may be doneexcept for the last part of the hydrolysis and thus (most of) the oxygenis consumed before the hydrolysed biomass enters the fermentationreactor. Advantageously the oxygen, preferably in the form of (gaseous)bubbles, is added in the last part of the hydrolysis step.

The inventors pose the hypothesis that in the first part of the(enzymatic) hydrolysis (step) amorphous polysaccharides are hydrolysedto sugars such as glucose and that in the second part of the hydrolysisstep the remaining crystalline polysaccharides are converted to sugars.Amorphous polysaccharides are for example converted to oligosaccharidesby endoglucanases whereafter the oligosaccharides can be converted bycellobiohydrolase and beta-glucosidase (BG) to sugars. According to thepresent hypothesis amorphous polysaccharides are located on the outsideof polysaccharides or polysaccharide complexes whereas crystallinepolysaccharides are located relatively more in the inside of thepolysaccharides or polysaccharide complexes present in theligno-cellulosic material. So the conversion of the crystallinepolysaccharides may continue even when most of the amorphouspolypeptides are hydrolysed. Especially the addition of oxygen isbeneficial during the hydrolysis of the crystalline polysaccharides forexample in the degradation of the polysaccharides into oligosaccharides.According to this hypothesis oxygen addition is especially useful in thesecond part of the hydrolysis step. In general, a shorter time of oxygenaddition (or shorter second part of hydrolysis) is needed in case ofrelatively low amounts of crystalline polysaccharides in theligno-cellulosic material compared hydrolysis of ligno-cellulosicmaterial in which relatively higher amounts of crystallinepolysaccharides are present. The inventors also pose that the additionof oxygen is beneficial for the hydrolysis of crystallinepolysaccharides. Therefore the addition of oxygen is very usefulespecially in the phase wherein crystalline polysaccharides are attackedby enzymes. Outside this phase not adding of oxygen might be moreefficient. Therefore the oxygen supply may start only in the second partor second half of the hydrolysis. At the end of the hydrolysis when mostof the crystalline polysaccharides are degraded, the oxygen addition ispreferably stopped. In the last part of the second part or second halfof the hydrolysis, most of the polysaccharides are converted tooligosaccharides which during further breakdown to smaller sugars do notneed oxygen anymore. Therefore preferably less oxygen, compared to theoxygen addition during the aerated part of the time, is added to theligno-cellulosic material at the end of the hydrolysis process or morepreferably no oxygen is added to the ligno-cellulosic material at theend of the hydrolysis process. This hypothesis is only given as possibleexplanation of the effect noticed by the inventors and the presentinvention does not fall or stand with the correctness of this theory.

The inventors have also noticed that aeration during an enzymatichydrolysis process in the beginning of the hydrolysis process results inan increased glucose production during the hydrolysis.

In FIG. 3 the effect of aeration is shown. Compared to the non-aeratedhydrolysis (shown as “non-aerated” curve), an aeration at the start ofthe hydrolysis process (shown as “aeration 0-7 hours” curve) will resultin an immediate increase in glucose production and for example alreadyafter 24 hours of hydrolysis a glucose production will be found thatcorresponds to a glucose production without aeration of 60 hourshydrolysis under identical conditions (except for aeration). Compared tothe non-aerated hydrolysis, an aeration at the last part of thehydrolysis process (shown as “aeration 72-100 hours” curve) will resultin an immediate increase in glucose production after aeration and forexample already after 24 hours after the start of aeration (at 72 hours)in the hydrolysis process a glucose production increase of 30% will befound compared to the glucose production without aeration underidentical conditions (except for aeration). It is believed by theinventors that by using an aeration at the start as well as at the lastpart of the hydrolysis process (with in between the aeration intervals aperiod of no aeration) might increase glucose production whereby thisresults in an increase of glucose production that is larger than one ofthe two separate increases. The present explanation is given to guideand instruct the skilled person in the art to select the properconditions for the present hydrolysis process.

Several examples of (partial) aeration during the enzymatic hydrolysisprocess are given in the Examples to show the beneficial effect of thepresent invention. This beneficial effect is found for severalsubstrates or feedstocks and therefore believed to be present for thehydrolysis of all kind of substrates or feedstocks.

Several examples of enzyme compositions for the enzymatic hydrolysisprocess are given in the Examples to show the beneficial effect of thepresent invention. This beneficial effect is found for several enzymecompositions and therefore believed to be present for all kind ofhydrolysing enzyme compositions.

According to a preferred embodiment of the invention the part of thetime wherein less or preferably no oxygen is added is 10 to 80%,preferably 20 to 80%, more preferably 30 to 80% and most preferably 40to 80% of the total enzymatic hydrolysis time. According to a furtherpreferred embodiment of the invention the part of the time wherein moreoxygen is added is 2 to 80%, preferably 4 to 60%, more preferably 8 to50% and most preferably 10 to 50% of the total enzymatic hydrolysistime. In general the oxygen concentration in the liquid phase during thepart of the time wherein oxygen is added is at least 2 times, preferablyat least 4 times, more preferably at least 10 times the oxygenconcentration in the liquid phase during the part of the time whereinless or no oxygen is added.

To a further preferred embodiment of the invention during the part ofthe time wherein oxygen addition takes place in the liquid phase (byaeration or addition of oxygen), the oxygen concentration (DO) in theliquid phase wherein the ligno-cellulosic material is present during theenzymatic hydrolysis, is at least 0.001 mol/m³, preferably at least0.002 mol/m³, more preferably at least 0.003 mol/m³ and even morepreferably more than 0.01 mol/m³, for example more than 0.02 mol/m³ or0.03 mol/m³. In reactors of less than 1 m³ DO values of below 0.01mol/m³ or 0.02 mol/m³ will be obtained by slow stirring. Vigorous mixingor stirring at such scale introduces part of the gas phase of theheadspace into the reaction liquid. For example the mixing or stirringmay create a whirlpool that draws oxygen into the liquid. In generalflushing the headspace with oxygen (for example in the form of air) incombination with (vigorous) mixing or stirring will introduce sufficientoxygen into the cellulosic material in the hydrolysis reactor forreactors up to a size of 100 liter to 1 m³. At larger scale, for examplein a reactor of 50 m³ or more, for example 100 m³, so much energy isneeded for vigorous stirring that from economic point of view this willnot be applied in a commercially operating process. In general in largereactors, stirring or mixing without introducing air or oxygen willresult in DO values of less than 0.01 mol/m³.

To still another preferred embodiment of the invention during the oxygengeneration or production the oxygen concentration in the liquid phase(aeration or addition of oxygen), the oxygen concentration in the liquidphase wherein the ligno-cellulosic material is present during theenzymatic hydrolysis, is during the part of the time wherein oxygen isadded preferably at most 80% of the saturation concentration of oxygenunder the hydrolysis reaction conditions, more preferably at most 0.12mol/m³, still more preferably at most 0.09 mol/m³, even more preferablyat most 0.06 mol/m³, even still more preferably at most 0.045 mol/m³ andmost preferably at most 0.03 mol/m³. Temperature and pressure willinfluence the DO. The preferred and exemplary mol/m³ values given aboverelate to normal atmospheric pressure and a temperature of about 62° C.The skilled person in the art will appreciate favourable DO values onbasis of the present teachings.

To a further preferred embodiment of the invention the oxygenconcentration in the liquid phase, wherein the ligno-cellulosic materialis present during the enzymatic hydrolysis, is during the part of thetime wherein less or no oxygen is added less than 0.02 mol/m³,preferably less than 0.01 mol/m³, more preferably less than 0.005mol/m³, and most preferably less than 0.001 mol/m³.

The oxygen addition in the form of air or other oxygen-containing gasaccording to the invention may also be used to at least partially stiror mix the hydrolysis reactor contents. The present process of theinvention shows especially on pilot plant and industrial scaleadvantages. Preferably the hydrolysis reactor has a volume of 1 m³ ormore, preferably of more than 10 m³ and most preferably of 50 m³ ormore. In general the hydrolysis reactor will be smaller than 3000 m³ or5000 m³ The inventors pose the theory that especially at large scaleinsufficient oxygen is available for the hydrolysis which might be dueto oxygen transfer limitations in the reactor for example in thecellulolytic biomass. On lab-scale experiments this oxygen-insufficiencymay play a less important role. The surface area (or oxygen contact areaof the reactor content) to reactor volume ratio is more favourable forsmall scale experiments than in large scale experiments. Moreover mixingin small scale experiments is relatively easier than at large scale.During those small scale experiments also the transport of oxygen fromthe headspace of the hydrolysis reactor is faster than compared to thesituation in large scale experiments. This theory is only given aspossible explanation of the effect noticed by the inventors, and thepresent invention does not fall or stands with the correctness of thistheory. According to a further embodiment of the invention the additionof oxygen may be used to control at least partially the hydrolysisprocess.

On large scale (for example in a reactor having a volume of more than 1m³ it was noticed that the oxygen addition resulted in improvedhydrolysis and/or a faster hydrolysis. Moreover the inventors were ableto further improve the hydrolysis process by using smaller amounts ofenzymes (on protein) and still obtaining high hydrolysis levels. One ofthe major costs of the hydrolysis is the cost of the enzyme or enzymecomposition. Therefore reducing the amount of enzymes needed is anenormous economic advantage.

The process of the present invention makes it possible to use amounts ofenzyme (or enzyme composition) of less than 7.5 mg enzyme/g glucan,preferably less than 6.0 mg enzyme/g glucan, more preferably less than5.0 mg glucan/g feedstock, still more preferably less than 4.0 mgenzyme/g glucan and most preferably less than 2.5 mg enzyme/g glucan (ondry matter and enzyme as protein).

The glucan content of acid treated feedstock is about 25 to 50 wt % (drymatter). For corn stover the glucan content is about 30 to 45 wt % (drymatter).

Therefore in general amounts of enzyme (or enzyme composition) ofbetween 0.1 and 7.5 mg enzyme/g glucan, preferably between 0.2 and 7.5mg enzyme/g glucan, more preferably of between 0.2 and 6.0 mg enzyme/gglucan, even more preferably of between 0.2 and 5.0 mg enzyme/g glucan,still more preferably of between 0.2 and 4.0 mg enzyme/g glucan and mostpreferably of between 0.2 and 2.5 mg enzyme/g glucan (on dry matter andenzyme as protein) will be used in the process of the invention.

The process of the present invention makes it possible to use amounts ofenzyme (or enzyme composition) of less than 3.0 mg enzyme/g feedstock,preferably less than 2.5 mg enzyme/g feedstock, more preferably lessthan 2.0 mg enzyme/g feedstock, still more preferably less than 1.5 mgenzyme/g feedstock and most preferably less than 1.0 mg enzyme/gfeedstock (on dry matter and enzyme as protein).

In general amounts of enzyme (or enzyme composition) of between 0.05 and3.0 mg enzyme/g feedstock, preferably between 0.1 and 3.0 mg enzyme/gfeedstock, more preferably of between 0.1 and 2.5 mg enzyme/g feedstock,even more preferably of between 0.1 and 2.0 mg enzyme/g feedstock, stillmore preferably of between 0.1 and 1.5 mg enzyme/g feedstock and mostpreferably of between 0.1 and 1.0 mg enzyme/g feedstock (on dry matterand enzyme as protein) will be used in the process of the invention.

By using these amounts of enzyme (or enzyme composition), a glucanconversion level of more 70% can be obtained, preferably more than 72%.In general glucan conversion levels of between 70% and 90% can beobtained, preferably levels of between 72% and 90% and even morepreferably levels of between 74% and 90%. The glucan conversion leveldetermination is described in the Examples.

The process of the invention is advantageously applied in combinationwith the use of thermostable enzymes.

A “thermostable” enzyme means that the enzyme has a temperature optimum60° C. or higher, for example 70° C. or higher, such as 75° C. orhigher, for example 80° C. or higher such as 85° C. or higher. They mayfor example be isolated from thermophilic microorganisms, or may bedesigned by the skilled person and artificially synthesized. In oneembodiment the polynucleotides may be isolated or obtained fromthermophilic or thermotolerant filamentous fungi or isolated fromnon-thermophilic or non-thermotolerant fungi but are found to bethermostable.

By “thermophilic fungus” is meant a fungus that grows at a temperatureof 50° C. or above. By “thermotolerant” fungus is meant a fungus thatgrows at a temperature of 45° C. or above, having a maximum near 50° C.

Examples of thermophilic fungal strains are Rasamsonia emersonii(formerly known as Talaromyces emersoni; Talaromyces emersonii,Penicillium Geosmithia emersonii and Rasamsonia emersonii are usedinterchangeably herein).

Suitable thermophilic or thermotolerant fungal cells may be a Humicola,Rhizomucor, Myceliophthora, Rasamsonia, Talaromyces, Thermomyces,Thermoascus or Thielavia cell, preferably a Rasamsonia emersonii cell.Preferred thermophilic or thermotolerant fungi are Humicola grisea var.thermoidea, Humicola lanuginosa, Myceliophthora thermophila, Papulasporathermophilia, Rasamsonia byssochlamydoides, Rasamsonia emersonii,Rasamsonia argillacea, Rasamsonia eburnean, Rasamsonia brevistipitata,Rasamsonia cylindrospora, Rhizomucor pusillus, Rhizomucor miehei,Talaromyces baciffisporus, Talaromyces leycettanus, Talaromycesthermophilus, Thermomyces lenuginosus, Thermoascus crustaceus,Thermoascus thermophilus Thermoascus aurantiacus and Thielaviaterrestris.

Thermophilic fungi are not restricted to a specific taxonomic order andoccur all over the fungal tree of life. Examples are Rhizomucor in theMucorales, Myceliophthora in Sordariales and Talaromyces, Thermomycesand Thermoascus in the Eurotiales (Mouchacca 1997). The majority ofTalaromyces species are mesophiles but exceptions are species withinsections Emersonii and Thermophila. Section Emersonii includesTalaromyces emersonii, Talaromyces byssochlamydoides, Talaromycesbaciffisporus and Talaromyces leycettanus, all of which grow well at 40°C. Talaromyces baciffisporus is thermotolerant, T. leycettanus isthermotolerant to thermophilic, and T. emersonii and T.byssochlamydoides are truly thermophilic (Stolk and Samson 1972). Thesole member of Talaromyces section Thermophila, T. thermophilus, growsrapidly at 50° C. (Evans and Stolk 1971; Evans 1971; Stolk and Samson1972). The current classification of these thermophilic Talaromycesspecies is mainly based on phenotypic and physiological characters, suchas their ability to grow above 40° C., ascospore color, the structure ofascornatal covering and the formation of a certain type of anamorph.Stolk and Samson (1972) stated that the members of the section Emersoniihave anamorphs of either Paecilomyces (T. byssochlamydoides and T.leycettanus) or Penicillium cylindrosporum series (T. emersonii and T.baciffisporus). Later, Pitt (1979) transferred the species belonging tothe Penicillium cylindrosporum series to the genus Geosmithia, based onvarious characters such as the formation of conidia from terminal poresinstead of on collula (necks), a character of Penicillium andPaecilomyces. Within the genus Geosmithia, only G. argillacea isthermotolerant, and Stolk et al. (1969) and Evans (1971) proposed aconnection with members of Talaromyces sect. Emersonii. The phylogeneticrelationship of the themophilic Talaromyces species within Talaromycesand the Trichocomaceae is unknown. See J. Houbraken, Antonie vanLeeuwenhoek 2012 February; 101(2): 403-21.

Rasamsonia is a new genus comprising thermotolerant and thermophilicTalaromyces and Geosmithia species (J. Houbraken et al vida supra).Based on phenotypic, physiological and molecular data, Houbraken et alproposed to transfer the species T. emersonii, T. byssochlamydoides, T.eburneus, G. argillacea and G. cylindrospora to Rasamsonia gen. nov.Talaromyces emersonii, Penicillium Geosmithia emersonii and Rasamsoniaemersonii are used interchangeably herein.

Preferred thermophilic fungi are Rasamsonia byssochlamydoides,Rasamsonia emersonii, Thermomyces lenuginosus, Talaromyces thermophilus,Thermoascus crustaceus, Thermoascus thermophilus and Thermoascusaurantiacus.

“Filamentous fungi” include all filamentous forms of the subdivisionEumycota and Oomycota (as defined by Hawksworth et al., In, Ainsworthand Bisby's Dictionary of The Fungi, 8th edition, 1995, CABInternational, University Press, Cambridge, UK). The filamentous fungiare characterized by a mycelial wall composed of chitin, cellulose,glucan, chitosan, mannan, and other complex polysaccharides. Vegetativegrowth is by hyphal elongation and carbon catabolism is obligatelyaerobic. Filamentous fungal strains include, but are not limited to,strains of Acremonium, Agaricus, Aspergillus, Aureobasidium,Chrysosporium, Coprinus, Cryptococcus, Filibasidium, Fusarium,Geosmithia, Humicola, Magnaporthe, Mucor, Myceliophthora,Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces,Panerochaete, Pleurotus, Rasamsonia, Schizophyllum, Talaromyces,Thermoascus, Thermomyces, Thielavia, Tolypocladium, and Trichoderma.

Several strains of filamentous fungi are readily accessible to thepublic in a number of culture collections, such as the American TypeCulture Collection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), andAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL). Examples of such strains includeAspergillus niger CBS 513.88, Aspergillus oryzae ATCC 20423, IFO 4177,ATCC 1011, ATCC 9576, ATCC14488-14491, ATCC 11601, ATCC12892, P.chrysogenum CBS 455.95, Penicillium citrinum ATCC 38065, Penicilliumchrysogenum P2, Talaromyces emersonii CBS 393.64, Acremonium chrysogenumATCC 36225 or ATCC 48272, Trichoderma reesei ATCC 26921 or ATCC 56765 orATCC 26921, Aspergillus sojae ATCC11906, Chrysosporium lucknowense C1,Garg 27K, VKM F-3500-D, ATCC44006 and derivatives thereof.

An advantage of expression and production of the enzymes (for example atleast two, three or four different cellulases) in a suitablemicroorganism may be a high enzyme composition yield which can be usedin the process of the present invention.

According to the invention, by the addition of oxygen it is possible toattain many process advantages, including optimal temperatureconditions, reduced process time, reduced dosage of enzyme, re-use ofenzymes and other process optimizations, resulting in reduced costs.Advantageously the invention provides a process in which the hydrolysisstep is conducted at improved conditions. The invention also provides aprocess involving hydrolysis having a reduced process time. Furthermorethe invention provides a process, wherein the dosage of enzyme may bereduced and at the same time output of useful hydrolysis product ismaintained at the same level. Another advantage of the invention is thatthe present process involving hydrolysis may result in processconditions which are optimized. A still further advantage of theinvention is that the output of useful hydrolysis product of the processinvolving hydrolysis is increased using the same enzyme dosage.

Stable Enzyme Composition

Stable enzyme composition herein means that the enzyme compositionretains activity after 30 hours of hydrolysis reaction time, preferablyat least 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80% 85%, 90%, 95%,96%, 97%, 98%, 99% or 100% of its initial activity after 30 hours ofhydrolysis reaction time. Preferably the enzyme composition retainsactivity after 40, 50, 60, 70, 80, 90 100, 150, 200, 250, 300, 350, 400,450, 500 hours of hydrolysis reaction time.

The enzyme composition may be prepared by fermentation of a suitablesubstrate with a suitable microorganism, e.g. Rasamsonia emersonii orAspergillus niger wherein the enzyme composition is produced by themicroorganism. The microorganism may be altered to improve or to makethe cellulase composition. For example the microorganism may be mutatedby classical strain improvement procedures or by recombinant DNAtechniques. Therefore the microorganisms mentioned herein can be used assuch to produce the cellulase composition or may be altered to increasethe production or to produce an altered cellulase composition whichmight include heterologous cellulases, thus enzymes that are notoriginally produced by that microorganism. Preferably a fungus, morepreferably a filamentous fungus is used to produce the cellulasecomposition. Advantageously a thermophilic or thermotolerantmicroorganism is used. Optionally a substrate is used that induces theexpression of the enzymes in the enzyme composition during theproduction of the enzyme composition.

The enzyme composition is used to release sugars from lignocellulose,that comprises polysaccharides. The major polysaccharides are cellulose(glucans), hemicelluloses (xylans, heteroxylans and xyloglucans). Inaddition, some hemicellulose may be present as glucomannans, for examplein wood-derived feedstocks. The enzymatic hydrolysis of thesepolysaccharides to soluble sugars, including both monomers andmultimers, for example glucose, cellobiose, xylose, arabinose,galactose, fructose, mannose, rhamnose, ribose, galacturonic acid,glucoronic acid and other hexoses and pentoses occurs under the actionof different enzymes acting in concert. By sugar product is meant theenzymatic hydrolysis product of the feedstock or ligno-cellulosicmaterial. The sugar product will comprise soluble sugars, including bothmonomers and multimers, preferably will comprise glucose. Examples ofother sugars are cellobiose, xylose, arabinose, galactose, fructose,mannose, rhamnose, ribose, galacturonic acid, glucoronic acid and otherhexoses and pentoses. The sugar product may be used as such or may befurther processed for example purified.

In addition, pectins and other pectic substances such as arabinans maymake up considerably proportion of the dry mass of typically cell wallsfrom non-woody plant tissues (about a quarter to half of dry mass may bepectins).

Cellulose is a linear polysaccharide composed of glucose residues linkedby β-1,4 bonds. The linear nature of the cellulose fibers, as well asthe stoichiometry of the β-linked glucose (relative to α) generatesstructures more prone to interstrand hydrogen bonding than the highlybranched α-linked structures of starch. Thus, cellulose polymers aregenerally less soluble, and form more tightly bound fibers than thefibers found in starch.

Enzymes that may be included in the stable enzyme composition used inthe invention are now described in more detail:

GH61, Endoglucanases (EG) and exo-cellobiohydrolases (CBH) catalyze thehydrolysis of insoluble cellulose to products such ascellooligosaccharides (cellobiose as a main product), whileβ-glucosidases (BG) convert the oligosaccharides, mainly cellobiose andcellotriose to glucose.

Hemicellulose is a complex polymer, and its composition often varieswidely from organism to organism and from one tissue type to another. Ingeneral, a main component of hemicellulose is β-1,4-linked xylose, afive carbon sugar. However, this xylose is often branched at 0 to 3and/or 0 to 2 atom of xylose, and can be substituted with linkages toarabinose, galactose, mannose, glucuronic acid, galacturonic acid or byesterification to acetic acid (and esterification of ferulic acid toarabinose). Hemicellulose can also contain glucan, which is a generalterm for β-linked six carbon sugars (such as the β-(1,3)(1,4) glucansand heteroglucans mentioned previously) and additionally glucomannans(in which both glucose and mannose are present in the linear backbone,linked to each other by β-linkages).

Xylanases together with other accessory enzymes, for exampleα-L-arabinofuranosidases, feruloyl and acetylxylan esterases,glucuronidases, and β-xylosidases) catalyze the hydrolysis ofhemicelluloses.

Pectic substances include pectins, arabinans, galactans andarabinogalactans. Pectins are the most complex polysaccharides in theplant cell wall. They are built up around a core chain of α(1,4)-linkedD-galacturonic acid units interspersed to some degree with L-rhamnose.In any one cell wall there are a number of structural units that fitthis description and it has generally been considered that in a singlepectic molecule, the core chains of different structural units arecontinuous with one another.

The principal types of structural unit are: galacturonan(homogalacturonan), which may be substituted with methanol on thecarboxyl group and acetate on O-2 and O-3; rhamnogalacturonan I (RGI),in which galacturonic acid units alternate with rhamnose units carrying(1,4)-linked galactan and (1,5)-linked arabinan side-chains. Thearabinan side-chains may be attached directly to rhamnose or indirectlythrough the galactan chains; xylogalacturonan, with single xylosyl unitson O-3 of galacturonic acid (closely associated with RGI); andrhamnogalacturonan II (RGII), a particularly complex minor unitcontaining unusual sugars, for example apiose. An RGII unit may containtwo apiosyl residues which, under suitable ionic conditions, canreversibly form esters with borate.

A composition for use in a method of the invention comprises preferablyat least two activities, although typically a composition will comprisemore than two activities, for example, three, four, five, six, seven,eight, nine or more. Typically, a composition of the invention maycomprise at least two different cellulases or one cellulase and at leastone hemicellulase. A composition of the invention may comprisecellulases, but no xylanases. In addition, a composition of theinvention may comprise auxiliary enzyme activity, i.e. additionalactivity which, either directly or indirectly leads to lignocellulosedegradation. Examples of such auxiliary activities are mentioned herein.

Thus, a composition for use in the invention may comprise GH61,endoglucanase activity and/or cellobiohydrolase activity and/orβ-glucosidase activity. A composition for use in the invention maycomprise more than one enzyme activity in one or more of those classes.For example, a composition for use in the invention may comprise twoendoglucanase activities, for example, endo-1,3(1,4)-β glucanaseactivity and endo-β-1,4-glucanase activity. Such a composition may alsocomprise one or more xylanase activities. Such a composition maycomprise an auxiliary enzyme activity.

A composition for use in the invention may be derived from Rasamsoniaemersonii. In the invention, it is anticipated that a core set of(lignocellulose degrading) enzyme activities may be derived fromRasamsonia emersonii. Rasamsonia emersonii can provide a highlyeffective set of activities as demonstrated herein for the hydrolysis oflignocellulosic biomass. That activity can then be supplemented withadditional enzyme activities from other sources. Such additionalactivities may be derived from classical sources and/or produced by agenetically modified organism.

The activities in a composition for use in the invention may bethermostable. Herein, this means that the activity has a temperatureoptimum of about 60° C. or higher, for example about 70° C. or higher,such as about 75° C. or higher, for example about 80° C. or higher suchas 85° C. or higher. Activities in a composition for use in theinvention will typically not have the same temperature optima, butpreferably will, nevertheless, be thermostable.

In addition, enzyme activities in a composition for use in the inventionmay be able to work at low pH. For the purposes of this invention, lowpH indicates a pH of about 5.5 or lower, about 5 or lower, about 4.9 orlower, about 4.8 or lower, about 4.7 or lower, about 4,6 or lower, about4.5 or lower, about 4.4 or lower, about 4.3 or lower, about 4.2 orlower, about 4,1 or lower, about 4.0 or lower about 3.9 or lower, orabout 3.8 or lower, about 3.7 or lower, about 3.6 or lower, or about 3.5or lower.

Activities in a composition for use in the invention may be defined by acombination of any of the above temperature optima and pH values.

The composition used in a method of the invention may comprise, inaddition to the activities derived from Rasamsonia, a cellulase (forexample one derived from a source other than Rasamsonia) and/or ahemicellulase (for example one derived from a source other thanRasamsonia) and/or a pectinase.

A composition for use in the invention may comprise one, two, three,four classes or more of cellulase, for example one, two three or four orall of a GH61, an endoglucanase (EG), one or two exo-cellobiohydrolase(CBH) and a β-glucosidase (BG). A composition for use in the inventionmay comprise two or more of any of these classes of cellulase.

A composition of the invention may comprise an activity which has adifferent type of cellulase activity and/or hemicellulase activityand/or pectinase activity than that provided by the composition for usein a method of the invention. For example, a composition of theinvention may comprise one type of cellulase and/or hemicellulaseactivity and/or pectinase activity provided by a composition asdescribed herein and a second type of cellulase and/or hemicellulaseactivity and/or pectinase activity provided by an additionalcellulose/hemicellulase/pectinase.

Herein, a cellulase is any polypeptide which is capable of degrading ormodifying cellulose. A polypeptide which is capable of degradingcellulose is one which is capable of catalysing the process of breakingdown cellulose into smaller units, either partially, for example intocellodextrins, or completely into glucose monomers. A cellulaseaccording to the invention may give rise to a mixed population ofcellodextrins and glucose monomers when contacted with the cellulase.Such degradation will typically take place by way of a hydrolysisreaction.

GH61 (glycoside hydrolase family 61 or sometimes referred to EGIV)proteins are oxygen-dependent polysaccharide monooxygenases (PMO's)according to the latest literature. Often in literature these proteinsare mentioned to enhance the action of cellulases on lignocellulosesubstrates. GH61 was originally classified as endogluconase based onmeasurement of very weak endo-1,4-β-D-dlucanase activity in one familymember. The term “GH61” as used herein, is to be understood as a familyof enzymes, which share common conserved sequence portions and foldingsto be classified in family 61 of the well-established CAZY GHclassification system (.cazy.org/GH61.html). The glycoside hydrolasefamily 61 is a member of the family of glycoside hydrolases EC 3.2.1.GH61 is used herein as being part of the cellulases.

Herein, a hemicellulase is any polypeptide which is capable of degradingor modifying hemicellulose. That is to say, a hemicellulase may becapable of degrading or modifying one or more of xylan, glucuronoxylan,arabinoxylan, glucomannan and xyloglucan. A polypeptide which is capableof degrading a hemicellulose is one which is capable of catalysing theprocess of breaking down the hemicellulose into smaller polysaccharides,either partially, for example into oligosaccharides, or completely intosugar monomers, for example hexose or pentose sugar monomers. Ahemicellulase according to the invention may give rise to a mixedpopulation of oligosaccharides and sugar monomers when contacted withthe hemicellulase. Such degradation will typically take place by way ofa hydrolysis reaction.

Herein, a pectinase is any polypeptide which is capable of degrading ormodifying pectin. A polypeptide which is capable of degrading pectin isone which is capable of catalysing the process of breaking down pectininto smaller units, either partially, for example into oligosaccharides,or completely into sugar monomers. A pectinase according to theinvention may give rise to a mixed population of oligosacchardies andsugar monomers when contacted with the pectinase. Such degradation willtypically take place by way of a hydrolysis reaction.

Accordingly, a composition of the invention may comprise any cellulase,for example, a GH61, a cellobiohydrolase, an endo-β-1,4-glucanase, aβ-glucosidase or a β-(1,3)(1,4)-glucanase.

Herein, a cellobiohydrolase (EC 3.2.1.91) is any polypeptide which iscapable of catalysing the hydrolysis of 1,4-β-D-glucosidic linkages incellulose or cellotetraose, releasing cellobiose from the ends of thechains. This enzyme may also be referred to as cellulase1,4-β-cellobiosidase, 1,4-β-cellobiohydrolase, 1,4-β-D-glucancellobiohydrolase, avicelase, exo-1,4-β-D-glucanase,exocellobiohydrolase or exoglucanase.

Herein, an endo-β-1,4-glucanase (EC 3.2.1.4) is any polypeptide which iscapable of catalysing the endohydrolysis of 1,4-β-D-glucosidic linkagesin cellulose, lichenin or cereal β-D-glucans. Such a polypeptide mayalso be capable of hydrolyzing 1,4-linkages in β-D-glucans alsocontaining 1,3-linkages. This enzyme may also be referred to ascellulase, avicelase, β-1,4-endoglucan hydrolase, β-1,4-glucanase,carboxymethyl cellulase, celludextrinase, endo-1,4-β-D-glucanase,endo-1,4-β-D-glucanohydrolase, endo-1,4-β-glucanase or endoglucanase.

Herein, a β-glucosidase (EC 3.2.1.21) is any polypeptide which iscapable of catalysing the hydrolysis of terminal, non-reducingβ-D-glucose residues with release of β-D-glucose. Such a polypeptide mayhave a wide specificity for β-D-glucosides and may also hydrolyze one ormore of the following: a β-D-galactoside, an α-L-arabinoside, aβ-D-xyloside or a β-D-fucoside. This enzyme may also be referred to asamygdalase, β-D-glucoside glucohydrolase, cellobiase or gentobiase.

Herein a β-(1,3)(1,4)-glucanase (EC 3.2.1.73) is any polypeptide whichis capable of catalyzing the hydrolysis of 1,4-β-D-glucosidic linkagesin β-D-glucans containing 1,3- and 1,4-bonds. Such a polypeptide may acton lichenin and cereal β-D-glucans, but not on β-D-glucans containingonly 1,3- or 1,4-bonds. This enzyme may also be referred to aslicheninase, 1,3-1,4-β-D-glucan 4-glucanohydrolase, β-glucanase,endo-β-1,3-1,4 glucanase, lichenase or mixed linkage β-glucanase. Analternative for this type of enzyme is EC 3.2.1.6, which is described asendo-1,3(4)-beta-glucanase. This type of enzyme hydrolyses 1,3- or1,4-linkages in beta-D-glucanse when the glucose residue whose reducinggroup is involved in the linkage to be hydrolysed is itself substitutedat C-3. Alternative names include endo-1,3-beta-glucanase, laminarinase,1,3-(1,3;1,4)-beta-D-glucan 3 (4) glucanohydrolase; substrates includelaminarin, lichenin and cereal beta-D-glucans.

A composition of the invention may comprise any hemicellulase, forexample, an endoxylanase, a β-xylosidase, a α-L-arabionofuranosidase, anα-D-glucuronidase, an acetyl xylan esterase, a feruloyl esterase, acoumaroyl esterase, an α-galactosidase, a β-galactosidase, a β-mannanaseor a β-mannosidase.

Herein, an endoxylanase (EC 3.2.1.8) is any polypeptide which is capableof catalyzing the endohydrolysis of 1,4-β-D-xylosidic linkages inxylans. This enzyme may also be referred to as endo-1,4-β-xylanase or1,4-β-D-xylan xylanohydrolase. An alternative is EC 3.2.1.136, aglucuronoarabinoxylan endoxylanase, an enzyme that is able to hydrolyse1,4 xylosidic linkages in glucuronoarabinoxylans.

Herein, a β-xylosidase (EC 3.2.1.37) is any polypeptide which is capableof catalyzing the hydrolysis of 1,4-β-D-xylans, to remove successiveD-xylose residues from the non-reducing termini. Such enzymes may alsohydrolyze xylobiose. This enzyme may also be referred to as xylan1,4-β-xylosidase, 1,4-β-D-xylan xylohydrolase, exo-1,4-β-xylosidase orxylobiase.

Herein, an α-L-arabinofuranosidase (EC 3.2.1.55) is any polypeptidewhich is capable of acting on α-L-arabinofuranosides, α-L-arabinanscontaining (1,2) and/or (1,3)- and/or (1,5)-linkages, arabinoxylans andarabinogalactans. This enzyme may also be referred to asα-N-arabinofuranosidase, arabinofuranosidase or arabinosidase.

Herein, an α-D-glucuronidase (EC 3.2.1.139) is any polypeptide which iscapable of catalyzing a reaction of the following form:alpha-D-glucuronoside+H(2)O=an alcohol+D-glucuronate. This enzyme mayalso be referred to as alpha-glucuronidase or alpha-glucosiduronase.These enzymes may also hydrolyse 4-O-methylated glucoronic acid, whichcan also be present as a substituent in xylans. Alternative is EC3.2.1.131: xylan alpha-1,2-glucuronosidase, which catalyses thehydrolysis of alpha-1,2-(4-O-methyl)glucuronosyl links.

Herein, an acetyl xylan esterase (EC 3.1.1.72) is any polypeptide whichis capable of catalyzing the deacetylation of xylans andxylo-oligosaccharides. Such a polypeptide may catalyze the hydrolysis ofacetyl groups from polymeric xylan, acetylated xylose, acetylatedglucose, alpha-napthyl acetate or p-nitrophenyl acetate but, typically,not from triacetylglycerol. Such a polypeptide typically does not act onacetylated mannan or pectin.

Herein, a feruloyl esterase (EC 3.1.1.73) is any polypeptide which iscapable of catalyzing a reaction of the form:feruloyl-saccharide+H(2)O=ferulate+saccharide. The saccharide may be,for example, an oligosaccharide or a polysaccharide. It may typicallycatalyze the hydrolysis of the 4-hydroxy-3-methoxycinnamoyl (feruloyl)group from an esterified sugar, which is usually arabinose in ‘natural’substrates. p-nitrophenol acetate and methyl ferulate are typicallypoorer substrates. This enzyme may also be referred to as cinnamoylester hydrolase, ferulic acid esterase or hydroxycinnamoyl esterase. Itmay also be referred to as a hemicellulase accessory enzyme, since itmay help xylanases and pectinases to break down plant cell wallhemicellulose and pectin.

Herein, a coumaroyl esterase (EC 3.1.1.73) is any polypeptide which iscapable of catalyzing a reaction of the form:coumaroyl-saccharide+H(2)O=coumarate+saccharide. The saccharide may be,for example, an oligosaccharide or a polysaccharide. This enzyme mayalso be referred to as trans-4-coumaroyl esterase, trans-p-coumaroylesterase, p-coumaroyl esterase or p-coumaric acid esterase. This enzymealso falls within EC 3.1.1.73 so may also be referred to as a feruloylesterase.

Herein, an α-galactosidase (EC 3.2.1.22) is any polypeptide which iscapable of catalyzing the hydrolysis of of terminal, non-reducingα-D-galactose residues in α-D-galactosides, including galactoseoligosaccharides, galactomannans, galactans and arabinogalactans. Such apolypeptide may also be capable of hydrolyzing α-D-fucosides. Thisenzyme may also be referred to as melibiase.

Herein, a β-galactosidase (EC 3.2.1.23) is any polypeptide which iscapable of catalyzing the hydrolysis of terminal non-reducingβ-D-galactose residues in β-D-galactosides. Such a polypeptide may alsobe capable of hydrolyzing α-L-arabinosides. This enzyme may also bereferred to as exo-(1→4)-β-D-galactanase or lactase.

Herein, a β-mannanase (EC 3.2.1.78) is any polypeptide which is capableof catalyzing the random hydrolysis of 1,4-β-D-mannosidic linkages inmannans, galactomannans and glucomannans. This enzyme may also bereferred to as mannan endo-1,4-β-mannosidase or endo-1,4-mannanase.

Herein, a β-mannosidase (EC 3.2.1.25) is any polypeptide which iscapable of catalyzing the hydrolysis of terminal, non-reducingβ-D-mannose residues in β-D-mannosides. This enzyme may also be referredto as mannanase or mannase.

A composition of the invention may comprise any pectinase, for examplean endo polygalacturonase, a pectin methyl esterase, anendo-galactanase, a beta galactosidase, a pectin acetyl esterase, anendo-pectin lyase, pectate lyase, alpha rhamnosidase, anexo-galacturonase, an expolygalacturonate lyase, a rhamnogalacturonanhydrolase, a rhamnogalacturonan lyase, a rhamnogalacturonan acetylesterase, a rhamnogalacturonan galacturonohydrolase, axylogalacturonase.

Herein, an endo-polygalacturonase (EC 3.2.1.15) is any polypeptide whichis capable of catalyzing the random hydrolysis of1,4-α-D-galactosiduronic linkages in pectate and other galacturonans.This enzyme may also be referred to as polygalacturonase pectindepolymerase, pectinase, endopolygalacturonase, pectolase, pectinhydrolase, pectin polygalacturonase, poly-α-1,4-galacturonideglycanohydrolase, endogalacturonase; endo-D-galacturonase orpoly(1,4-α-D-galacturonide) glycanohydrolase.

Herein, a pectin methyl esterase (EC 3.1.1.11) is any enzyme which iscapable of catalyzing the reaction: pectin+n H₂O=n methanol+pectate. Theenzyme may also been known as pectinesterase, pectin demethoxylase,pectin methoxylase, pectin methylesterase, pectase, pectinoesterase orpectin pectylhydrolase.

Herein, an endo-galactanase (EC 3.2.1.89) is any enzyme capable ofcatalyzing the endohydrolysis of 1,4-β-D-galactosidic linkages inarabinogalactans. The enzyme may also be known as arabinogalactanendo-1,4-β-galactosidase, endo-1,4-β-galactanase, galactanase,arabinogalactanase or arabinogalactan 4-β-D-galactanohydrolase.

Herein, a pectin acetyl esterase is defined herein as any enzyme whichhas an acetyl esterase activity which catalyzes the deacetylation of theacetyl groups at the hydroxyl groups of GalUA residues of pectin

Herein, an endo-pectin lyase (EC 4.2.2.10) is any enzyme capable ofcatalyzing the eliminative cleavage of (1→4)-α-D-galacturonan methylester to give oligosaccharides with4-deoxy-6-O-methyl-α-D-galact-4-enuronosyl groups at their non-reducingends. The enzyme may also be known as pectin lyase, pectintrans-eliminase; endo-pectin lyase, polymethylgalacturonictranseliminase, pectin methyltranseliminase, pectolyase, PL, PNL or PMGLor (1→4)-6-O-methyl-α-D-galacturonan lyase.

Herein, a pectate lyase (EC 4.2.2.2) is any enzyme capable of catalyzingthe eliminative cleavage of (1→4)-α-D-galacturonan to giveoligosaccharides with 4-deoxy-α-D-galact-4-enuronosyl groups at theirnon-reducing ends. The enzyme may also be known polygalacturonictranseliminase, pectic acid transeliminase, polygalacturonate lyase,endopectin methyltranseliminase, pectate transeliminase,endogalacturonate transeliminase, pectic acid lyase, pectic lyase,α-1,4-D-endopolygalacturonic acid lyase, PGA lyase, PPase-N,endo-α-1,4-polygalacturonic acid lyase, polygalacturonic acid lyase,pectin trans-eliminase, polygalacturonic acid trans-eliminase or(1→4)-α-D-galacturonan lyase.

Herein, an alpha rhamnosidase (EC 3.2.1.40) is any polypeptide which iscapable of catalyzing the hydrolysis of terminal non-reducingα-L-rhamnose residues in α-L-rhamnosides or alternatively inrhamnogalacturonan. This enzyme may also be known as α-L-rhamnosidase T,α-L-rhamnosidase N or α-L-rhamnoside rhamnohydrolase.

Herein, exo-galacturonase (EC 3.2.1.82) is any polypeptide capable ofhydrolysis of pectic acid from the non-reducing end, releasingdigalacturonate. The enzyme may also be known asexo-poly-α-galacturonosidase, exopolygalacturonosidase orexopolygalacturanosidase.

Herein, exo-galacturonase (EC 3.2.1.67) is any polypeptide capable ofcatalyzing:(1,4-α-D-galacturonide)_(n)+H₂O=(1,4-α-D-galacturonide)_(n-1)+D-galacturonate.The enzyme may also be known as galacturan 1,4-α-galacturonidase,exopolygalacturonase, poly(galacturonate) hydrolase,exo-D-galacturonase, exo-D-galacturonanase, exopoly-D-galacturonase orpoly(1,4-α-D-galacturonide) galacturonohydrolase.

Herein, exopolygalacturonate lyase (EC 4.2.2.9) is any polypeptidecapable of catalyzing eliminative cleavage of4-(4-deoxy-α-D-galact-4-enuronosyl)-D-galacturonate from the reducingend of pectate, i.e. de-esterified pectin. This enzyme may be known aspectate disaccharide-lyase, pectate exo-lyase, exopectic acidtranseliminase, exopectate lyase, exopolygalacturonicacid-trans-eliminase, PATE, exo-PATE, exo-PGL or (1→4)-α-D-galacturonanreducing-end-disaccharide-lyase.

Herein, rhamnogalacturonan hydrolase is any polypeptide which is capableof hydrolyzing the linkage between galactosyluronic acid acid andrhamnopyranosyl in an endo-fashion in strictly alternatingrhamnogalacturonan structures, consisting of the disaccharide[(1,2-alpha-L-rhamnoyl-(1,4)-alpha-galactosyluronic acid].

Herein, rhamnogalacturonan lyase is any polypeptide which is anypolypeptide which is capable of cleaving α-L-Rhap-(1→4)-α-D-GalpAlinkages in an endo-fashion in rhamnogalacturonan by beta-elimination.

Herein, rhamnogalacturonan acetyl esterase is any polypeptide whichcatalyzes the deacetylation of the backbone of alternating rhamnose andgalacturonic acid residues in rhamnogalacturonan.

Herein, rhamnogalacturonan galacturonohydrolase is any polypeptide whichis capable of hydrolyzing galacturonic acid from the non-reducing end ofstrictly alternating rhamnogalacturonan structures in an exo-fashion.

Herein, xylogalacturonase is any polypeptide which acts onxylogalacturonan by cleaving the β-xylose substituted galacturonic acidbackbone in an endo-manner. This enzyme may also be known asxylogalacturonan hydrolase.

Herein, an α-L-arabinofuranosidase (EC 3.2.1.55) is any polypeptidewhich is capable of acting on α-L-arabinofuranosides, α-L-arabinanscontaining (1,2) and/or (1,3)- and/or (1,5)-linkages, arabinoxylans andarabinogalactans. This enzyme may also be referred to asα-N-arabinofuranosidase, arabinofuranosidase or arabinosidase.

Herein, endo-arabinanase (EC 3.2.1.99) is any polypeptide which iscapable of catalyzing endohydrolysis of 1,5-α-arabinofuranosidiclinkages in 1,5-arabinans. The enzyme may also be know asendo-arabinase, arabinan endo-1,5-α-L-arabinosidase,endo-1,5-α-L-arabinanase, endo-α-1,5-arabanase; endo-arabanase or1,5-α-L-arabinan 1,5-α-L-arabinanohydrolase.

A composition of the invention will typically comprise at least onecellulase and/or at least one hemicellulase and/or at least onepectinase (one of which is a polypeptide according to the invention). Acomposition of the invention may comprise a GH61, a cellobiohydrolase,an endoglucanase and/or a β-glucosidase. Such a composition may alsocomprise one or more hemicellulases and/or one or more pectinases.

In addition, one or more (for example two, three, four or all) of anamylase, a protease, a lipase, a ligninase, a hexosyltransferase, aglucuronidase or an expansin or a cellulose induced protein or acellulose integrating protein or like protein may be present in acomposition of the invention (these are referred to as auxiliaryactivities above).

“Protease” includes enzymes that hydrolyze peptide bonds (peptidases),as well as enzymes that hydrolyze bonds between peptides and othermoieties, such as sugars (glycopeptidases). Many proteases arecharacterized under EC 3.4, and are suitable for use in the inventionincorporated herein by reference. Some specific types of proteasesinclude, cysteine proteases including pepsin, papain and serineproteases including chymotrypsins, carboxypeptidases andmetalloendopeptidases.

“Lipase” includes enzymes that hydrolyze lipids, fatty acids, andacylglycerides, including phospoglycerides, lipoproteins,diacylglycerols, and the like. In plants, lipids are used as structuralcomponents to limit water loss and pathogen infection. These lipidsinclude waxes derived from fatty acids, as well as cutin and suberin.

“Ligninase” includes enzymes that can hydrolyze or break down thestructure of lignin polymers. Enzymes that can break down lignin includelignin peroxidases, manganese peroxidases, laccases and feruloylesterases, and other enzymes described in the art known to depolymerizeor otherwise break lignin polymers. Also included are enzymes capable ofhydrolyzing bonds formed between hemicellulosic sugars (notablyarabinose) and lignin. Ligninases include but are not limited to thefollowing group of enzymes: lignin peroxidases (EC 1.11.1.14), manganeseperoxidases (EC 1.11.1.13), laccases (EC 1.10.3.2) and feruloylesterases (EC 3.1.1.73).

“Hexosyltransferase” (2.4.1-) includes enzymes which are capable ofcatalyzing a transferase reaction, but which can also catalyze ahydrolysis reaction, for example of cellulose and/or cellulosedegradation products. An example of a hexosyltransferase which may beused in the invention is a β-glucanosyltransferase. Such an enzyme maybe able to catalyze degradation of (1,3)(1,4)glucan and/or celluloseand/or a cellulose degradation product.

“Glucuronidase” includes enzymes that catalyze the hydrolysis of aglucoronoside, for example β-glucuronoside to yield an alcohol. Manyglucuronidases have been characterized and may be suitable for use inthe invention, for example β-glucuronidase (EC 3.2.1.31),hyalurono-glucuronidase (EC 3.2.1.36), glucuronosyl-disulfoglucosamineglucuronidase (3.2.1.56), glycyrrhizinate β-glucuronidase (3.2.1.128) orα-D-glucuronidase (EC 3.2.1.139).

A composition for use in the invention may comprise an expansin orexpansin-like protein, such as a swollenin (see Salheimo et al., Eur. J.Biohem. 269, 4202-4211, 2002) or a swollenin-like protein.

Expansins are implicated in loosening of the cell wall structure duringplant cell growth. Expansins have been proposed to disrupt hydrogenbonding between cellulose and other cell wall polysaccharides withouthaving hydrolytic activity. In this way, they are thought to allow thesliding of cellulose fibers and enlargement of the cell wall. Swollenin,an expansin-like protein contains an N-terminal Carbohydrate BindingModule Family 1 domain (CBD) and a C-terminal expansin-like domain. Forthe purposes of this invention, an expansin-like protein orswollenin-like protein may comprise one or both of such domains and/ormay disrupt the structure of cell walls (such as disrupting cellulosestructure), optionally without producing detectable amounts of reducingsugars.

A composition for use in the invention may be a cellulose inducedprotein, for example the polypeptide product of the dpi or cip2 gene orsimilar genes (see Foreman et al., J. Biol. Chem. 278(34), 31988-31997,2003), a cellulose/cellulosome integrating protein, for example thepolypeptide product of the cipA or cipC gene, or a scaffoldin or ascaffoldin-like protein. Scaffoldins and cellulose integrating proteinsare multi-functional integrating subunits which may organizecellulolytic subunits into a multi-enzyme complex. This is accomplishedby the interaction of two complementary classes of domain, i.e. acohesion domain on scaffoldin and a dockerin domain on each enzymaticunit. The scaffoldin subunit also bears a cellulose-binding module (CBM)that mediates attachment of the cellulosome to its substrate. Ascaffoldin or cellulose integrating protein for the purposes of thisinvention may comprise one or both of such domains.

A composition for use in a method of the invention may be composed of amember of each of the classes of enzymes mentioned above, severalmembers of one enzyme class, or any combination of these enzymes classesor helper proteins (i.e. those proteins mentioned herein which do nothave enzymatic activity per se, but do nevertheless assist inlignocellulosic degradation).

A composition for use in a method of the invention may be composed ofenzymes from (1) commercial suppliers; (2) cloned genes expressingenzymes; (3) complex broth (such as that resulting from growth of amicrobial strain in media, wherein the strains secrete proteins andenzymes into the media; (4) cell lysates of strains grown as in (3);and/or (5) plant material expressing enzymes. Different enzymes in acomposition of the invention may be obtained from different sources.

The enzymes can be produced either exogenously in microorganisms,yeasts, fungi, bacteria or plants, then isolated and added, for example,to lignocellulosic feedstock. Alternatively, the enzymes are produced,but not isolated, and crude cell mass fermentation broth, or plantmaterial (such as corn stover or wheat straw), and the like may be addedto, for example, the feedstock. Alternatively, the crude cell mass orenzyme production medium or plant material may be treated to preventfurther microbial growth (for example, by heating or addition ofantimicrobial agents), then added to, for example, a feedstock. Thesecrude enzyme mixtures may include the organism producing the enzyme.Alternatively, the enzyme may be produced in a fermentation that uses(pre-treated) feedstock (such as corn stover or wheat straw) to providenutrition to an organism that produces an enzyme(s). In this manner,plants that produce the enzymes may themselves serve as alignocellulosic feedstock and be added into lignocellulosic feedstock.

In the uses and methods described herein, the components of thecompositions described above may be provided concomitantly (i.e. as asingle composition per se) or separately or sequentially.

The invention thus relates to methods in which the composition describedabove are used and to uses of the composition in industrial processes.

Limo-Cellulosic Material

Lignocellulosic material herein includes any lignocellulosic and/orhemicellulosic material. Lignocellulosic material suitable for use asfeedstock in the invention includes biomass, e.g. virgin biomass and/ornon-virgin biomass such as agricultural biomass, commercial organics,construction and demolition debris, municipal solid waste, waste paperand yard waste. Common forms of biomass include trees, shrubs andgrasses, wheat, wheat straw, sugar cane bagasse, switch grass,miscanthus, corn, corn stover, corn husks, corn cobs, canola stems,soybean stems, sweet sorghum, corn kernel including fiber from kernels,products and by-products from milling of grains such as corn, wheat andbarley (including wet milling and dry milling) often called “bran orfibre” as well as municipal solid waste, waste paper and yard waste. Thebiomass can also be, but is not limited to, herbaceous material,agricultural residues, forestry residues, municipal solid wastes, wastepaper, and pulp and paper mill residues. “Agricultural biomass” includesbranches, bushes, canes, corn and corn husks, energy crops, forests,fruits, flowers, grains, grasses, herbaceous crops, leaves, bark,needles, logs, roots, saplings, short rotation woody crops, shrubs,switch grasses, trees, vegetables, fruit peels, vines, sugar beet pulp,wheat midlings, oat hulls, and hard and soft woods (not including woodswith deleterious materials). In addition, agricultural biomass includesorganic waste materials generated from agricultural processes includingfarming and forestry activities, specifically including forestry woodwaste. Agricultural biomass may be any of the aforementioned singularlyor in any combination or mixture thereof.

Pre-Treatment

The feedstock may optionally be pre-treated with heat, mechanical and/orchemical modification or any combination of such methods in order to toenhance the accessibility of the substrate to enzymatic hydrolysisand/or hydrolyse the hemicellulose and/or solubilize the hemicelluloseand/or cellulose and/or lignin, in any way known in the art. In oneembodiment, the pre-treatment is conducted treating the lignocellulosewith steam explosion, hot water treatment or treatment with dilute acidor dilute base.

Washing Step

Optionally, the process according to the invention comprises a washingstep. The optional washing step may be used to remove water solublecompounds that may act as inhibitors for the fermentation step. Thewashing step may be conducted in known manner.

Enzymatic Hydrolysis

The enzyme composition used in the process of the invention canextremely effectively hydrolyze lignocellulolytic material, for examplecorn stover or wheat straw, which can then be further converted into auseful product, such as ethanol, biogas, butanol, lactic acid, aplastic, an organic acid, a solvent, an animal feed supplement, apharmaceutical, a vitamin, an amino acid, an enzyme or a chemicalfeedstock. Additionally, intermediate products from a process followingthe hydrolysis, for example lactic acid as intermediate in biogasproduction, can be used as building block for other materials. Thepresent invention is exemplified with the production of ethanol but thisis done as exemplification only rather than as limitation, the othermentioned useful products can be produced equally well.

The process according to the invention comprises an enzymatic hydrolysisstep. The enzymatic hydrolysis includes, but is not limited to,hydrolysis for the purpose of liquification of the feedstock andhydrolysis for the purpose of releasing sugar from the feedstock orboth. In this step optionally pre-treated and optionally washedligno-cellulosic material is brought into contact with the enzymecomposition according to the invention. Depending on the lignocellulosicmaterial and the pre-treatment, the different reaction conditions, e.g.temperature, enzyme dosage, hydrolysis reaction time and dry matterconcentration, may be adapted by the skilled person in order to achievea desired conversion of lignocellulose to sugar. Some indications aregiven hereafter.

In one aspect of the invention the hydrolysis is conducted at atemperature of 45° C. or more, of 50° C. or more, 55° C. or more, 60° C.or more, 65° C. or more, or 70° C. or more. The high temperature duringhydrolysis has many advantages, which include working at the optimumtemperature of the enzyme composition, the reduction of risk of(bacterial) contamination, reduced viscosity, smaller amount of coolingwater required, use of cooling water with a higher temperature, re-useof the enzymes and more.

In a further aspect of the invention, the amount of enzyme compositionadded (herein also called enzyme dosage or enzyme load) is low. In anembodiment the amount of enzyme is 6 mg protein/g dry matter weight orlower, 5 mg protein/g dry matter or lower, 4 mg protein/g dry matter orlower, 3 mg protein/g dry matter or lower, 2 mg protein/g dry matter orlower, or 1 mg protein/g dry matter or lower (expressed as protein in mgprotein/g dry matter). In an embodiment, the amount of enzyme is 0.5 mgenzyme/g dry matter weight or lower, 0.4 mg enzyme composition/g drymatter weight or lower, 0.3 mg enzyme/g dry matter weight or lower, 0.25mg enzyme/g dry matter weight or lower, 0.20 mg enzyme/g dry matterweight or lower, 0.18 mg enzyme/g dry matter weight or lower, 0.15 mgenzyme/g dry matter weight or lower or 0.10 mg enzyme/g dry matterweight or lower (expressed as total of cellulase enzymes in mg enzyme/gdry matter). Low enzyme dosage is possible, since because of theactivity and stability of the enzymes, it is possible to increase thehydrolysis reaction time.

In a further aspect of the invention, the hydrolysis reaction time is 5hours or more, 10 hours or more, 20 hours or more, 40 hours or more, 50hours or more, 60 hours or more, 70 hours or more, 80 hours or more, 90hours or more, 100 hours or more, 120 hours or more, 130 h or more. Inanother aspect, the hydrolysis reaction time is 5 to 150 hours, 40 to130 hours, 50 to 120 hours, 60 to 120 hours, 60 to 110 hours, 60 to 100hours, 70 to 100 hours, 70 to 90 hours or 70 to 80 hours. Due to thestability of the enzyme composition longer hydrolysis reaction times arepossible with corresponding higher sugar yields.

The pH during hydrolysis may be chosen by the skilled person. In afurther aspect of the invention, the pH during the hydrolysis may be 3.0to 6.4. The stable enzymes of the invention may have a broad pH range ofup to 2 pH units, up to 3 pH units, up to 5 pH units. The optimum pH maylie within the limits of pH 2.0 to 8.0, 3.0 to 8.0, 3.5 to 7.0, 3.5 to6.0, 3.5 to 5.0, 3.5 to 4.5, 4.0 to 4.5 or is about 4.2.

In a further aspect of the invention the hydrolysis step is conducteduntil 70% or more, 80% or more, 85% or more, 90% or more, 92% or more,95% or more of available sugar in lignocellulosic material is released.

Significantly, a process of the invention may be carried out using highlevels of dry matter (of the lignocellulosic material) in the hydrolysisreaction. Thus, the invention may be carried out with a dry mattercontent of about 5 wt % or higher, about 8 wt % or higher, about 10 wt %or higher, about 11 wt % or higher, about 12 wt % or higher, about 13 wt% or higher, about 14 wt % or higher, about 15 wt % or higher, about 20wt % or higher, about 25 wt % or higher, about 30 wt % or higher, about35 wt % or higher or about 40 wt % or higher. In a further embodiment,the dry matter content in the hydrolysis step is 14 wt %, 15 wt %, 16 wt%, 17 wt %, 18 wt %, 19 wt %, 20 wt %, 21 wt %, 22 wt %, 23 wt %, 24 wt%, 25 wt %, 26 wt %, 27 wt %, 28 wt %, 29 wt %, 30 wt %, 31 wt %, 32 wt%, 33 wt % or more or 14 to 33 wt %.

Fermentation

The process according to the invention comprises a fermentation step. Ina further aspect, the invention thus includes in step fermentationprocesses in which a microorganism is used for the fermentation of acarbon source comprising sugar(s), e.g. glucose, L-arabinose and/orxylose. The carbon source may include any carbohydrate oligo- or polymercomprising L-arabinose, xylose or glucose units, such as e.g.lignocellulose, xylans, cellulose, starch, arabinan and the like. Forrelease of xylose or glucose units from such carbohydrates, appropriatecarbohydrases (such as xylanases, glucanases, amylases and the like) maybe added to the fermentation medium or may be produced by the modifiedhost cell. In the latter case the modified host cell may be geneticallyengineered to produce and excrete such carbohydrases. An additionaladvantage of using oligo- or polymeric sources of glucose is that itenables to maintain a low(er) concentration of free glucose during thefermentation, e.g. by using rate-limiting amounts of the carbohydrases.This, in turn, will prevent repression of systems required formetabolism and transport of non-glucose sugars such as xylose. In apreferred process the modified host cell ferments both the L-arabinose(optionally xylose) and glucose, preferably simultaneously in which casepreferably a modified host cell is used which is insensitive to glucoserepression to prevent diauxic growth. In addition to a source ofL-arabinose, optionally xylose (and glucose) as carbon source, thefermentation medium will further comprise the appropriate ingredientrequired for growth of the modified host cell. Compositions offermentation media for growth of microorganisms such as yeasts orfilamentous fungi are well known in the art.

The fermentation time may be shorter than in conventional fermentationat the same conditions, wherein part of the enzymatic hydrolysis stillhas to take part during fermentation. In one embodiment, thefermentation time is 100 hours or less, 90 hours or less, 80 hours orless, 70 hours or less, or 60 hours or less, for a sugar composition of50 g/l glucose and corresponding other sugars from the lignocellulosicfeedstock (e.g. 50 g/l xylose, 35 g/l L-arabinose and 10 g/l galactose.For more dilute sugar compositions the fermentation time maycorrespondingly be reduced.

The fermentation process may be an aerobic or an anaerobic fermentationprocess. An anaerobic fermentation process is herein defined as afermentation process run in the absence of oxygen or in whichsubstantially no oxygen is consumed, preferably less than 5, 2.5 or 1mmol/L/h, more preferably 0 mmol/L/h is consumed (i.e. oxygenconsumption is not detectable), and wherein organic molecules serve asboth electron donor and electron acceptors. In the absence of oxygen,NADH produced in glycolysis and biomass formation, cannot be oxidised byoxidative phosphorylation. To solve this problem many microorganisms usepyruvate or one of its derivatives as an electron and hydrogen acceptorthereby regenerating NAD⁺. Thus, in a preferred anaerobic fermentationprocess pyruvate is used as an electron (and hydrogen acceptor) and isreduced to fermentation products such as ethanol, lactic acid,3-hydroxy-propionic acid, acrylic acid, acetic acid, succinic acid,citric acid, malic acid, fumaric acid, an amino acid, 1,3-propane-diol,ethylene, glycerol, butanol, a β-lactam antibiotics and a cephalosporin.In a preferred embodiment, the fermentation process is anaerobic. Ananaerobic process is advantageous since it is cheaper than aerobicprocesses: less special equipment is needed. Furthermore, anaerobicprocesses are expected to give a higher product yield than aerobicprocesses. Under aerobic conditions, usually the biomass yield is higherthan under anaerobic conditions. As a consequence, usually under aerobicconditions, the expected product yield is lower than under anaerobicconditions.

In another embodiment, the fermentation process is under oxygen-limitedconditions. More preferably, the fermentation process is aerobic andunder oxygen-limited conditions. An oxygen-limited fermentation processis a process in which the oxygen consumption is limited by the oxygentransfer from the gas to the liquid. The degree of oxygen limitation isdetermined by the amount and composition of the ingoing gas flow as wellas the actual mixing/mass transfer properties of the fermentationequipment used. Preferably, in a process under oxygen-limitedconditions, the rate of oxygen consumption is at least 5.5, morepreferably at least 6 and even more preferably at least 7 mmol/L/h.

The fermentation process is preferably run at a temperature that isoptimal for the modified cell. Thus, for most yeasts or fungal cells,the fermentation process is performed at a temperature which is lessthan 42° C., preferably less than 38° C. For yeast or filamentous fungalhost cells, the fermentation process is preferably performed at atemperature which is lower than 35, 33, 30 or 28° C. and at atemperature which is higher than 20, 22, or 25° C.

In an embodiment of the invention, in step the fermentation is conductedwith a microorganism that is able to ferment at least one C5 sugar. Inan embodiment the process is a process for the production of ethanolwhereby the process comprises the step comprises fermenting a mediumcontaining sugar(s) with a microorganism that is able to ferment atleast one C5 sugar, whereby the host cell is able to ferment glucose,L-arabinose and xylose to ethanol. In an embodiment thereof themicroorganism that is able to ferment at least one C5 sugar is a yeast.In an embodiment, the yeast is belongs to the genus Saccharomyces,preferably of the species Saccharomyces cerevisiae, in which geneticmodifications have been made. An example of such a microorganism and itspreparation is described in more detail in WO 2008/041840 and inEuropean Patent Application EP10160622.6, filed 21 Apr. 2010. In anembodiment, the fermentation process for the production of ethanol isanaerobic. Anaerobic has already been defined earlier herein. In anotherpreferred embodiment, the fermentation process for the production ofethanol is aerobic. In another preferred embodiment, the fermentationprocess for the production of ethanol is under oxygen-limitedconditions, more preferably aerobic and under oxygen-limited conditions.Oxygen-limited conditions have already been defined earlier herein.

In such process, the volumetric ethanol productivity is preferably atleast 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 5.0 or 10.0 g ethanol per litre perhour. The ethanol yield on L-arabinose and optionally xylose and/orglucose in the process preferably is at least 20, 25, 30, 35, 40, 45,50, 60, 70, 80, 90, 95 or 98%. The ethanol yield is herein defined as apercentage of the theoretical maximum yield, which, for glucose andL-arabinose and optionally xylose is 0.51 g. ethanol per g. glucose orxylose.

In one aspect, the fermentation process leading to the production ofethanol, has several advantages by comparison to known ethanolfermentations processes:

-   -   anaerobic processes are possible;    -   oxygen limited conditions are also possible;    -   higher ethanol yields and ethanol production rates can be        obtained;    -   the strain used may be able to use L-arabinose and optionally        xylose.

Alternatively to the fermentation processes described above, at leasttwo distinct cells may be used, this means this process is aco-fermentation process. All preferred embodiments of the fermentationprocesses as described above are also preferred embodiments of thisco-fermentation process: identity of the fermentation product, identityof source of L-arabinose and source of xylose, conditions offermentation (aerobical or anaerobical conditions, oxygen-limitedconditions, temperature at which the process is being carried out,productivity of ethanol, yield of ethanol).

The fermentation process may be carried out without any requirement toadjust the pH during the process. That is to say, the process is onewhich may be carried out without the addition of any acid(s) or base(s).However, this excludes a pretreatment step, where acid may be added. Thepoint is that the composition of the invention is capable of acting atlow pH and, therefore, there is no need to adjust the pH of acid of anacid pretreated feedstock in order that saccharification or hydrolysismay take place. Accordingly, a method of the invention may be a zerowaste method using only organic products with no requirement forinorganic chemical input.

Overall Reaction Time

According to the invention, the overall reaction time (or the reactiontime of hydrolysis step and fermentation step together) may be reduced.In one embodiment, the overall reaction time is 300 hours or less, 200hours or less, 150 hours or less, 140 hours or less, 130 or less, 120hours or less, 110 hours or less, 100 hours of less, 90 hours or less,80 hours or less, 75 hours or less, or about 72 hours at 90% glucoseyield. Correspondingly lower overall times may be reached at lowerglucose yield.

Fermentation Products

Fermentation products which may be produced according to the inventioninclude amino acids, vitamins, pharmaceuticals, animal feed supplements,specialty chemicals, chemical feedstocks, plastics, solvents, fuels, orother organic polymers, lactic acid, and ethanol, including fuel ethanol(the term “ethanol” being understood to include ethyl alcohol ormixtures of ethyl alcohol and water).

Specific value-added products that may be produced by the methods of theinvention include, but not limited to, biofuels (including biogas,ethanol and butanol); lactic acid; 3-hydroxy-propionic acid; acrylicacid; acetic acid; 1,3-propane-diol; ethylene; glycerol; a plastic; aspecialty chemical; an organic acid, including citric acid, succinicacid and maleic acid; a solvent; an animal feed supplement; apharmaceutical such as a β-lactam antibiotic or a cephalosporin; avitamin; an amino acid, such as lysine, methionine, tryptophan,threonine, and aspartic acid; an enzyme, such as a protease, acellulase, an amylase, a glucanase, a lactase, a lipase, a lyase, anoxidoreductase, a transferase or a xylanase; a chemical feedstock; or ananimal feed supplement.

Separation of Fermentation Product

The process according to the invention optionally comprises recovery offermentation product. A fermentation product may be separated from thefermentation broth in any known manner. For each fermentation productthe skilled person will thus be able to select a proper separationtechnique. For instance ethanol may be separated from a yeastfermentation broth by distillation, for instance steamdistillation/vacuum distillation in conventional way.

Certain embodiments of the invention will below be described in moredetail, but are in no way limiting the scope of the present invention.

Use of Thermostable Enzymes Under Optimal Temperature Conditions

In one embodiment, the invention relates to the use of thermostableenzymes such as cellulolytic enzymes of Rasamsonia for the production ofreducing sugars from pre-treated ligno-cellulosic feedstock in, but notlimiting to, ethanol production. Cellulolytic enzymes of Rasamsoniaapplied on pre-treated ligno-cellulosic feedstock showed maximalconversion rates at temperature within the range of 50 to 70° C. Theenzyme remains active under these circumstances for 14 days and morewithout complete cessation of activity.

By using optimal temperature conditions, maximal amount of reducingsugars can be released from feedstock (total hydrolysis) within theshortest possible hydrolysis time. In this way, 100% conversion ofcellulose in glucose is achieved in less than 5 days.

The theoretical maximum yield (Yps max in g product per gram glucose) ofa fermentation product can be derived from textbook biochemistry. Forethanol, 1 mole of glucose (180 g) yields according to normal glycolysisfermentation pathway in yeast 2 moles of ethanol (=2×46=92 g ethanol.The theoretical maximum yield of ethanol on glucose is therefore92/180=0.511 g ethanol/g glucose.

For butanol (MW 74 g/mole) or iso butanol, the theoretical maximum yieldis 1 mole of butanol per mole of glucose. So Yps max for(iso-)butanol=74/180=0.411 g (iso-)butanol/g glucose.

For lactic acid the fermentation yield for homolactic fermentation is 2moles of lactic acid (MW=90 g/mole) per mole of glucose. According tothis stoichiometry, the Yps max=1 g lactic acid/g glucose.

For other fermentation products a similar calculation may be made.

The cost reduction achieved with applying cellulolytic enzymes ofRasamsonia will be the result of an overall process time reduction.

Compensation of Lower Enzyme Dosage With Extended Hydrolysis Time UsingRasamsonia Enzymes

Due to the high stability of the stable enzymes, the activities do notcease in time, although less reducing sugars are liberated in the courseof the hydrolysis. It is possible to lower the enzyme dosage and extendthe use of the enzyme by prolonging the hydrolysis times to obtainsimilar levels of released reducing sugars. For example, 0.175 mLenzyme/g feedstock dry-matter resulted in release of approximately 90%of the theoretical maximum of reducing sugars from pre-treated feedstockwithin 72 h. When using 0.075 mL enzyme/g feedstock dry-matter,approximately 90% conversion of the theoretical maximum is achievedwithin 120 h. The results show that, because of the stability of theenzyme activity, lowering the enzyme dosage can be compensated byextending the hydrolysis time to obtain the same amount of reducingsugars. The same holds for hydrolysis of pre-treated feedstock atdry-matter contents higher than 10% shows that compensating effect ofextended hydrolysis time at 15% dry matter feedstock.

The cost reduction achieved by using stable cellulolytic enzymes, suchas of Rasamsonia, results from requiring less enzyme dosage, resultingin similar hydrolysis conversion yields.

Lowering the Risk on Contamination With Stable Enzymes

In a common process for converting ligno-cellulosic material intoethanol, process steps are preferably done under septic conditions tolower the operational costs. Contamination and growth of contaminatingmicroorganisms can therefore occur and result in undesirable sideeffects, such lactic acid, formic acid and acetic acid production, yieldlosses of ethanol on substrate, production of toxins and extracellularpolysaccharides, which may affect production costs significantly. A highprocess temperature and/or a short process time will limit the risk oncontamination during hydrolysis and fermentation. Thermostable enzymes,like those of Rasamsonia, are capable of hydrolysing ligno-cellulosicfeedstock at temperatures of higher than 60° C. At these temperatures,the risk that a contaminating microorganism will cause undesired sideeffects will be little to almost zero.

During the fermentation step, in which ethanol is produced, temperaturesare typically between 30 to 37° C. and will preferably not be raisedbecause of production losses. By applying fermentation process times asshort as possible the risks and effects of contamination and/or growthof contaminants will be reduced as much as possible. With stableenzymes, like those of Rasamsonia a short as possible fermentation timescan be applied (see description above), and thus risks on contaminationand/or growth of contaminants will be reduced as much as possible. Thecost reduction achieved with applying thermostable cellulolytic enzymesof Rasamsonia in this way will result from lower risk of processfailures due to contamination.

Stable Enzymes Reduce Cooling Costs and Increase Productivity of EthanolPlants

The first step after thermal pretreatment will be to cool the pretreatedfeedstock to temperatures where the enzymes are optimal active. On largescale, this is typically done by adding (cooled) water, which will,besides decreasing the temperature, reduce the dry-matter content. Byusing thermos stable enzymes, like those of Rasamsonia, cost reductioncan be achieved by the fact that (i) less cooling of the pretreatedfeedstock is required since higher temperatures are allowed duringhydrolysis, and (ii) less water will be added, which will increase thedry-matter content during hydrolysis and fermentation and thus increasethe ethanol production capacity (amount produced per time unit pervolume) of an ethanol plant. Also, by using thermostable enzymesaccording to the invention, like those of Rasamsonia, cost reduction mayalso be achieved by using cooling water having higher temperature thatthe water that is used in a process with non-thermostable enzyme.

Enzyme Recycling After Hydrolysis With Stable Enzymes

At the end of the hydrolysis, enzyme activities appear to be low sincelittle reducing sugars are released once almost all cellulose isconverted. The amount of enzymatic activity present, however, hasdecreased only a little, assumingly mainly due to absorption of theenzymes to the substrate. By applying solid-liquid separation afterhydrolysis, such as centrifugation, filtration, sedicantation, etcetera,60% or more e.g. 70% of the enzyme activity in solution can be recoveredand re-used for hydrolysis of a new pre-treated ligno-cellulosicfeedstock during the next hydrolysis.

Moreover, after solid-liquid separation the enzyme in solution can beseparated from the solution containing reducing sugars and otherhydrolysis products from the enzymatic actions. This separation can bedone by, but not limiting to, (ultra and micro)filtration,centrifugation, sedicantation, sedimentation, with or without firstadsorption of the enzyme to a carrier of any kind.

For example, after hydrolysis of pre-treated feedstock with 0.175 mL/gfeedstock dry matter enzyme load for 20 h, 50% of the theoreticalmaximum amount of reducing sugars is liberated and after the samehydrolysis for 72 h, 90% of the theoretical maximum amount of reducingsugars is liberated. By centrifugation and ultrafiltration, 60-70% ofthe enzyme activity was recovered in the retentate, while the filtratecontained more than 80% of the liberated reducing sugars. By re-usingthe retentate, either as it is or after further purification and/orconcentration, enzyme dosage during the next hydrolysis step can bereduced with 60 to 70%. The cost reduction achieved by using stablecellulolytic enzymes, such as of Rasamsonia, in this way results fromrequiring less enzyme dosage.

Enzyme Recycling After Hydrolysis in Combination With Enzyme Productionand Yeast-Cell Recycling With Stable Enzymes

The process including enzyme recycling after hydrolysis, as describedabove, can be combined with recycling of the ethanol producingmicroorganism after fermentation and with the use of the reducing sugarscontaining filtrate as a substrate (purified and/or concentrated ordiluted) in enzyme-production fermentation and as substrate for thecultivation of the ethanol-producing microorganism.

Enzyme Recycling After Vacuum Distillation With Stable Enzymes

The thermo stability of enzymes, like those from Rasamsonia, causesremaining cellulolytic activity after hydrolysis, fermentation andvacuum distillation in the thin stillage. The total activity of theenzyme is reduced during the three successive process steps. The thinstillage obtained after vacuum distillation can thus be re-used as asource of enzyme for a newly startedhydrolysis-fermentation-distillation process cycle of pre-treated wheatstraw conversion into ethanol. The thin stillage can be used either inconcentrated or (un)diluted form and/or purified and with or withoutadditional enzyme supplementation.

Enzyme Recycling in Combination With Enzyme Supplementation After VacuumDistillation With Thermostable Enzymes

In an optimal process, an amount of enzyme is supplemented into the thinstillage, before its re-use in a new process cycle, equal to the amountof activity lost during the three successive process steps of theprevious process cycle. In this way over-dosage of enzyme is avoided andthus most efficient use of enzyme is obtained.

Moreover, by providing high enzyme dosage in the first process cycle,and supplementing enzyme equal to the amount of activity lost during thethree successive process steps in the following process cycles, highestpossible hydrolysis rates can be obtained in each process cycleresulting in short hydrolysis times of less than 48 h in combinationwith most efficient use of enzymes.

Use of Stable Enzymes in Mixed Systems

By applying mixing during hydrolysis, enzymes come more often in contactwith substrates, which results in a more efficient use of the catalyticactivity. This will result in a lower enzyme dosages and thus in lowercosts, unless the mixing has a negative effect on the enzymes. Stableenzymes, like the thermostable enzymes from Rasamsonia, are robust andcan resist circumstances of (locally) high shear and temperatures, whichis the case during intensive mixing of slurries. The use of it in mixedsystems is therefore beneficial and will lead to dosage and thus costsreduction.

The invention is further described by the following examples, whichshould not be construed as limiting the scope of the invention.

Experimental Information

Strains

Rasamsonia (Talaromyces) emersonii strain was deposited at CENTRAALBUREAU VOOR SCHIMMELCULTURES, Uppsalalaan 8, P.O. Box 85167, NL-3508 ADUtrecht, The Netherlands in December 1964 having the Accession NumberCBS 393.64. Other suitable strains can be equally used in the presentexamples to show the effect and advantages of the invention. For exampleTEC-101, TEC-147, TEC-192, TEC-201 or TEC-210 are suitable Rasamsoniastrains which are described in WO2011/000949.

Preparation of Acid Pre-Treated Corn Stover Substrate.

Dilute-acid pre-treated corn stover (aCS) was obtained as described inSchell, D. J., Applied Biochemistry and Biotechnology (2003), vol.105-108, pp 69-85. A pilot scale pretreatment reactor was used operatingat steady state conditions of 190° C., 1 min residence time and aneffective H2SO4 acid concentration of 1.45% (w/w) in the liquid phase.

Protein Measurement Assays

1. Total Protein

TCA Biuret

The method was a combination of precipitation of protein using trichloroacetic acid (TCA) to remove disturbing substances and allowdetermination of the protein concentration with the colorimetric Biuretreaction. In the Biuret reaction, a copper (II) ion is reduced to copper(I), which forms a complex with the nitrogens and carbons of the peptidebonds in an alkaline solution. A violet color indicates the presence ofproteins. The intensity of the color, and hence the absorption at 546nm, is directly proportional to the protein concentration, according tothe Beer-Lambert law. The standardisation was performed using BSA(Bovine Serum Albumine) and the protein content was expressed in gprotein as BSA equivalent/L or mg protein as BSA equivalent/ml. Theprotein content was calculated using standard calculation protocolsknown in the art, by plotting the OD₅₄₆ versus the concentration ofsamples with known concentration, followed by the calculation of theconcentration of the unknown samples using the equation generated fromthe calibration line.

2. Individual Proteins Using PAGE

Sample Pre-Treatment SDS-PAGE

Based on the estimated protein concentration of the samples thefollowing samples preparation was performed. To 10 μl sample 40 μlMilliQ water and 50 μl TCA (20%) was added to dilute the sample fivetimes (˜1 mg/ml) and precipitate the proteins. After 1 hour on ice thesample was centrifuged (10 minutes, 14000 rpm). The pellet was washedwith 500 μl Aceton and centrifuged (10 minutes, 14000 rpm). The pelletwas treated as described below.

SDS-PAGE

The pellet was dissolved in 65 μl of the MilliQ water, 25 μl NuPAGE™ LDSsample buffer (4×) Invitrogen and 10 μl NuPAGE™ Sample Reducing agent(10×) Invitrogen. Prior to the the deanuarion step the sample wasdiluted 5 times using a mix of MilliQ; NuPAGE™ LDS sample buffer and 10μl NuPAGE™ Sample Reducing in the ratio of 65:25:10. After mixing, thesamples were incubated in a thermo mixer for 10 minutes at 70° C. Thesample solutions were applied on a 4-12% Bis-Tris gel (NuPAGE™ BisTris,Invitrogen). A sample (10 μl) of marker M12 (Invitrogen) was alsoapplied on the gel. The gel was run at 200 V for 50 minutes, using theXCELL Surelock, with 600 ml 20× diluted SDS buffer in the outer bufferchamber and 200 ml 20× diluted SDS buffer, containing 0.5 ml ofantioxidant (NuPAGE™ Invitrogen) in the inner buffer chamber. Afterrunning, the gel was rinsed twice with demineralised water the gels werefixed with 50% methanol/7% acetic acid solution for one hour and stainedwith Sypro Ruby (50 ml per gel) overnight. An image was made using theTyphoon 9200 (610 BP 30, Green (532 nm), PMT 600V, 100 micron) afterwashing the gel with MilliQ water.

Quantitative Analysis of the Protein

Using the Typhoon scanner the ratio between protein bands within a lanewas determined using standard methods known in the art. The sample wasapplied in triplicate and the gray values were determined using theprogram Image quant. Values are expressed as relative % protein to thetotal protein, calculated using the gray value of the selected proteinband relative to the total gray value all the protein bands.

Glucan Conversion Calculation:

-   glucan conversion (%)=(glucose (g/l)×100%)/(glucan (fraction on    DM)×dm (g/kg)×1.1)-   Wherein:-   glucose (g/l)=glucose concentration in supernatant after hydrolysis.-   glucan (fraction on dm)=glucan content of the substrate before    pretreatment.-   dm (g/kg)=dry matter of hydrolysis (f.i. 20% dm=200 g/kg).-   1.1=weight increase due to water incorporation during hydrolysis.

Example Calculation:

-   glucose=60 g/l-   glucan fraction=0.40 (is 40% on dry matter)-   dm=200 g/kg-   glucan conversion example=(60*100)/(0.4×200×1.1)=68% conversion

Example 1 Evaluation of the Effect of the Absence of Oxygen DuringHydrolysis on the Cellulolytic Activity of Cellulase Enzyme Cocktails

The effect of oxygen absence during hydrolysis on the cellulolyticactivity of three different enzyme cocktails was evaluated according tothe procedures described below. The hydrolysis reactions were performedwith acid pretreated cornstover (aCS) feedstock at a final concentrationof 10 w/w % DM. This feedstock solution was prepared via the dilution ofa concentrated feedstock solution with water. Subsequently the pH wasadjusted to pH 4.5 with a 4M NaOH solution. The elimination of oxygenfrom the feedstock was accomplished in two steps. First, the feedstocksolution was degassed via sonication under vacuum in a sonication bath(Bransonic 5510E-DTH, setting; Degas) for 15 minutes. In the secondstep, the oxygen was further removed by continuous sparging of anitrogen flow through a 500 ml solution of the 10% DM feedstock for aperiod of 3 hours. Prior to being sparged through the feedstocksolution, the nitrogen flow was sparged through water in order tosaturate it with water vapour and prevent evaporation of the water fromthe feedstock solution. In parallel, 500 ml of the same batch 10 w/w %DM aCS was sparged with air as an oxygen-containing control sample in asimilar set-up and according to the same protocol.

The hydrolysis of the oxygen-depleted (nitrogen sparged) and theoxygen-saturated (air-sparged) 10 w/w % aCS feedstock solutions wereconducted in air-tight, 30-ml centrifuge bottles (Nalgene Oakridge) in atotal reaction volume of 10 ml. The bottles, already containing thecellulase solution, used for the oxygen-depleted experiment were spargedwith nitrogen prior to and during filling them with feedstock. Eachhydrolysis was performed in duplicate with 7.5 mg/g DM cellulase enzymecocktail added in a total volume not larger than 375 μl. The threecellulase enzyme cocktails tested included: a TEC-210 mix (mixture ofcellulases), a 4E-GH61 mix (consisting of 9 w/w % of total protein BG,30 w/w % of total protein CBHI, 25 w/w % of total protein CBHII and 36w/w % of total protein GH61) and a 4E-EG mix (consisting of 9 w/w % oftotal protein BG, 30 w/w % of total protein CBHI, 25 w/w % of totalprotein CBHII and 36 w/w % of total protein EG). TEC-210 was fermentedaccording to the inoculation and fermentation procedures described inWO2011/000949. The 4E mix (as described in WO2011/098577) was used.

The centrifuge bottles containing the feedstock and enzyme solution wereplaced in an oven incubator (Techne HB-1D hybridization oven) andincubated for 72 hours at 65° C. while rotating at set-point 3 (12 rpmper minute). Following hydrolysis, the samples were cooled on ice andimmediately 50 μl of each supernatant was diluted in 1450 μl grade Iwater. The diluted supernatant was subsequently filtered (0.45 μmfilter, Pall PN 454) and the filtrates were analysed for sugar contentas described below.

The sugar concentrations of the diluted samples were measured using anHPLC equipped with an Aminex HPX-87P column (Biorad #1250098) by elutionwith water at 85° C. at a flow rate of 0.6 ml per minute and quantifiedby integration of the glucose signals from refractive index detection(R.I.) calibrated with glucose standard solutions.

The data presented in Table 1/FIG. 1 show that the glucose released fromthe nitrogen-sparged feedstocks is lower than the glucose released fromthe feedstocks sparged with air for both the TEC-210 mix and the 4E-GH61mix incubations. There is no difference in glucose release detectablebetween the nitrogen and air sparged feedstocks for samples hydrolyzedby the 4E-EG mix.

Based on these results we conclude that the presence of oxygen improvesthe cellulolytic performance of cellulase mixtures that contain GH61enzymes.

TABLE 1 The effect of sparging nitrogen or air through a 10% aCSfeedstock before hydrolysis, on the total amount of glucose released bythree different cellulase mixes. Sparged with air Sparged with N₂Cellulase Average glucose Average glucose cocktail (g/l) stdev (g/l)stdev TEC-210 34.5 0.8 31.9 1.1 4E-GH61 mix 31.7 1.4 27.4 0.1 4E-EG mix22.7 0.1 23.3 1.7

Example 2 The Effect of Oxygen on the Cellulolytic Activity of CellulaseEnzyme Cocktails During Hydrolysis of Lignocellulosic Feedstock

The effect of oxygen on the cellulolytic activity of the enzyme cocktailduring the hydrolysis of lignocellulosic feedstock is shown in thisexample. The hydrolysis reactions are performed with acid pretreatedcornstover (aCS) feedstock at a final concentration of 20 w/w % DM. Thisfeedstock solution is prepared via the dilution of a concentratedfeedstock solution with water. Subsequently the pH is adjusted to pH 4.5with a 10% (w/w) NH₄OH solution.

The hydrolysis is done in a stirred, pH controlled and temperaturecontrolled reactor with a working volume of 1 l. Each hydrolysis isperformed in duplicate with 2.5 mg/g DM TEC-210 cellulase enzymecocktail. TEC-210 was produced according to the inoculation andfermentation procedures described in WO2011/000949.

The following experiments are done:

-   1. 1 l of 20% aCS, pH 4.5, temperature 62° C., stirrer speed 60 rpm    (this corresponds with a DO level of <0.002 mol of oxygen per m³),    2.5 mg/g dm TEC-210 cellulase cocktail, incubation time 120 hours    (reference experiment).-   2. As experiment 1 but at the start of the hydrolysis, air sparging    into the solution started to a dissolved oxygen level of 20% (this    corresponds to 0.03 mol of oxygen per m3, measured using a DO    (dissolved oxygen) electrode). This dissolved oxygen level is    maintained throughout the rest of the hydrolysis process.-   3. As experiment 1 but at 72 hours air sparging into the solution    started to a dissolved oxygen level of 20% (this corresponds to 0.03    mol of oxygen per m3, measured using a DO (dissolved oxygen)    electrode). This dissolved oxygen level is maintained throughout the    rest of the hydrolysis process.

After the hydrolysis, the samples are cooled on ice and immediately 50μl of each supernatant is diluted in 1450 μl grade I water. The dilutedsupernatant is subsequently filtered (0.45 μm filter, Pall PN 454) andthe filtrates are analysed for sugar content as described below.

The sugar concentrations of the diluted samples are measured using anHPLC equipped with an Aminex HPX-87P column (Biorad #1250098) by elutionwith water at 85° C. at a flow rate of 0.6 ml per minute and quantifiedby integration of the glucose signals from refractive index detection(R.I.) calibrated with glucose standard solutions.

Results, visible in FIG. 2 clearly show an increased glucose productionin case air is added. In addition, air added to the hydrolysis reactionin the second part of the time demonstrates superior glucose productioncompared to no air addition or an air addition during the wholehydrolysis step.

Example 3 The Effect of Partial Aeration (in Time) on the EnzymaticHydrolysis of Lignocellulosic Feedstock on Pilot Scale

The effect of the dissolved oxygen concentration on the cellulolyticactivity of the enzyme cocktail or composition during the hydrolysis oflignocellulosic feedstock on pilot scale is shown in this example. Thehydrolysis reactions are performed with acid pretreated cornstover (aCS)feedstock at a final concentration of 20 w/w % DM. The feedstocksolution is prepared by the dilution of concentrated feedstock slurrywith water. The pH is adjusted to pH 4.5 with a 25% (w/w) NH₄OHsolution.

The enzymatic hydrolysis is done in a 270 liter pilot reactor which ispH and temperature controlled with a working volume of 150 liter. Thedissolved oxygen during the process is controlled by adjusting impellerspeed at a given airflow and overpressure. The enzymatic hydrolysis isperformed at a dosage of 2.5 mg (TCA protein)/g dm TEC-210 cellulaseenzyme cocktail. TEC-210 was produced according to the inoculation andfermentation procedures described in WO2011/000949.

The following experiments are done:

-   Experiment 1    -   Aeration from 0 to 120 hours: 150 l of 20% pCS, pH 4.5,        temperature 62° C., 1 bar overpressure, 10 kg/h airflow in the        headspace, 3.75 mg TCA/g dm TEC-210 cellulase cocktail,        incubation time 120 hours in a 270 liter pilot reactor The        dissolved oxygen concentration (DO) of the reaction mixture was        measured constantly using a DO electrode. The DO was controlled        at a level of 0.15-0.22 mol/m³ by adjusting the impeller speed.-   Experiment 2    -   Aeration between 72 and 120 hours: 150 l of 20% pCS, pH 4.5,        temperature 62° C., an enzyme dosage 2.50 mg TCA/g dm TEC-210        cellulase cocktail and a total incubation time of 120 hours in a        270 liter pilot reactor. The dissolved oxygen concentration (DO)        of the reaction mixture was measured constantly using a DO        electrode. For the first 72 hours of the process the following        settings were applied: no overpressure, no airflow in the        headspace and the DO was controlled at a level of [0.02-0.05]        mol/m3 by adjusting the impeller speed. For the last 48 hours of        the process the following settings were applied: 1 bar        overpressure, 10 kg/h airflow in the headspace and the DO was        controlled at a level of 0.15-0.22 mol/m³ by adjusting the        impeller speed.

During the enzymatic hydrolysis, samples were taken daily forcarbohydrate analysis (glucose, cellobiose) by NMR and viscosity and pHmeasurement.

Composition analysis of the pretreated Corn Stover was done by chemicalhydrolysis of the sample and determination of the mono saccharides byNMR.

Samples taken during enzymatic hydrolysis were analysed for(oligo)sugars, organic acids and inhibitors by flow NMR.

The results are presented in FIG. 4 and show that during enzymatichydrolysis in experiment 2 with the partial aeration (□=aeration betweenhydrolysis time is 72 and 100 hours) more glucose is produced thanduring enzymatic hydrolysis in experiment 1 (▪=aeration betweenhydrolysis time is 0 and 100 hours).

Example 4 The Effect of Timing of Dissolved Oxygen Supply on EnzymaticHydrolysis of Lignocellulosic Feedstock

The effect of timing of dissolved oxygen supply on the enzymatichydrolysis of lignocellulosic feedstock is shown in this example. Thehydrolysis reactions are performed with acid pretreated cornstover (aCS)feedstock at a final concentration of 20 w/w % DM. The feedstocksolution is prepared by the dilution of concentrated feedstock slurrywith water. The pH is adjusted to pH 4.5 with a 25% (w/w) NH₄OHsolution.

The enzymatic hydrolysis is done in a 2 liter reactor which is pH andtemperature controlled with a working volume of 1 liter. The dissolvedoxygen during the process is controlled by adjusting impeller speed andcontinuous refreshment of the headspace with fresh air in case of anincreased dissolved oxygen concentration. The enzymatic hydrolysis isperformed at a dosage of 1.5 mg (TCA protein)/g dm TEC-210 cellulaseenzyme cocktail. TEC-210 was produced according to the inoculation andfermentation procedures described in WO2011/000949.

The following experiments are done:

Experiment 1. Aeration from 0 to 7 hours: 1 l of 20% pCS, pH 4.5,temperature 62° C., 1.5 mg TCA/g dm TEC-210 cellulase cocktail,incubation time 120 hours. The dissolved oxygen concentration (DO) ofthe reaction mixture was measured constantly using a DO electrode. TheDO was controlled at a level of >0.05 mol/m³ during the first 7 hours ofthe hydrolysis process. Between 7 and 120 hours of hydrolysis time theDO was maintained at a level <0.02 mol/m³.

Experiment 2. Aeration between 72 and 120 hours: 1 l of 20% pCS, pH 4.5,temperature 62° C., 1.5 mg TCA/g dm TEC-210 cellulase cocktail,incubation time 120 hours. The dissolved oxygen concentration (DO) ofthe reaction mixture was measured constantly using a DO electrode. TheDO was controlled at a level of <0.01 mol/m³ during the first 72 hoursof the hydrolysis process. Between 72 and 120 hours of hydrolysis timethe DO was maintained at a level >0.05 mol/m³.

During the enzymatic hydrolysis, samples were taken daily forcarbohydrate analysis (glucose, cellobiose) by NMR and viscosity and pHmeasurement.

Composition analysis of the pretreated Corn Stover was done by chemicalhydrolysis of the sample and determination of the mono saccharides byNMR.

The results are presented in FIG. 5 and clearly demonstrate an increasein the glucose formation rate when the reaction mixture is aerated.Experiment 1, which was aerated between 0 and 7 hours, clearly shows anincreased glucose formation rate during the first 7 hours of the processcompared with the non-aerated situation during that process phase ofExperiment 2. In addition, Experiment 2 demonstrates an increasedglucose formation rate between 72 and 120 hours compared with thenon-aerated situation during that period in Experiment 1.

Example 5 The Effect of Oxygen on the Cellulolytic Activity of CellulaseEnzyme Compositions During Hydrolysis of Lignocellulosic Feedstock Usinga Low Enzyme Dosage

The effect of oxygen on the cellulolytic activity of the enzymecomposition using a low enzyme dosage during the hydrolysis oflignocellulosic feedstock is shown in this example. The hydrolysisreactions are performed with acid pretreated cornstover (aCS) feedstockat a final concentration of 20 w/w % DM. This feedstock solution isprepared via the dilution of a concentrated feedstock solution withwater. Subsequently the pH is adjusted to pH 4.5 with a 10% (w/w) NH₄OHsolution. The glucan content of the applied corn stover was 37% on drymatter.

The hydrolysis is done in a stirred, pH controlled and temperaturecontrolled reactor with a working volume of 1 l. Each hydrolysis isperformed in duplicate with 2.5 mg/g DM of TEC-210 cellulase enzymecomposition (or cocktail). TEC-210 was produced according to theinoculation and fermentation procedures described in WO2011/000949.

The following experiments are done:

-   -   1. 1 l of 20% aCS, pH 4.5, temperature 62° C., stirrer speed 60        rpm, 3.5 mg TEC-210 cellulase composition per gram feedstock (on        dry matter), incubation time 120 hours (reference experiment) in        a closed reactor. The dissolved oxygen level of the reaction        mixture was measured constantly using a DO electrode. This slow        stirring resulted in a dissolved oxygen level 0.005 mol/m³.    -   2. As experiment 1 but using and enzyme dosage of 2.5 mg TEC-210        per gram feedstock (on dry matter) and a stirrer speed of 250        rpm a head space over the reaction mixture which is constantly        refreshed with fresh air. The higher stirring speed in        combination with refreshment of the head space with fresh air        resulted in a dissolved oxygen level of. 0.030 mol/m³ in the        reaction mixture.

During hydrolysis samples were taken for analysis. The samples werecooled on ice and immediately 50 μl of each supernatant is diluted in1450 μl grade I water. The diluted supernatant is subsequently filtered(0.45 μm filter, Pall PN 454) and the filtrates are analysed for sugarcontent as described below.

The sugar concentrations of the diluted samples are measured using anHPLC equipped with an Aminex HPX-87P column (Biorad #1250098) by elutionwith water at 85° C. at a flow rate of 0.6 ml per minute and quantifiedby integration of the glucose signals from refractive index detection(R.I.) calibrated with glucose standard solutions.

The results are presented in FIG. 6.

The glucan conversions are listed in Table 2.

TABLE 2 glucan conversion levels. Enzyme dosage DO level Glucanconversion Experiment (mg/g dm) mol/m³ (%) 1 2.5 0.030 75 2 3.5 0.005 70

The invention claimed is:
 1. A process for preparation of a sugarproduct from lignocellulosic material, comprising: enzymatic hydrolysisof the lignocellulosic material using an enzyme composition comprisingat least two cellulases and whereby the enzyme composition at leastcomprises GH61; whereby between 0.1 and 7.5 mg enzyme composition/gglucan on dry matter and enzyme as protein or between 0.05 and 3.0 mgenzyme composition/g feedstock on dry matter and enzyme as protein isused; and wherein: the enzymatic hydrolysis occurs for a first part oftime and a subsequent second part of time; during the first part of timeless or no oxygen is added to the lignocellulosic material compared tothe second part of time, during the second part of time more oxygen isadded to the lignocellulosic material compared to the first part oftime, and the second part of time is 2% to 80% of total enzymatichydrolysis time.
 2. A process for preparation of a fermentation productfrom lignocellulosic material, comprising: enzymatic hydrolysis of thelignocellulosic material using an enzyme composition comprising at leasttwo cellulases and whereby the enzyme composition at least comprisesGH61; whereby between 0.1 and 7.5 mg enzyme composition/g glucan on drymatter and enzyme as protein or between 0.05 and 3.0 mg enzymecomposition/g feedstock on dry matter and enzyme as protein is used; andfermentation of the hydrolysed lignocellulosic material to produce afermentation product; and wherein: the enzymatic hydrolysis occurs for afirst part of time and a subsequent second part of time; during thefirst part of time less or no oxygen is added to the lignocellulosicmaterial compared to the second part of time, during the second part oftime more oxygen is added to the lignocellulosic material compared tothe first part of time, and the second part of time is 2% to 80% oftotal enzymatic hydrolysis time.
 3. The process according to claim 1,wherein the oxygen is added to the lignocellulosic material in the formof bubbles.
 4. The process according to claim 1, wherein the enzymatichydrolysis is performed in a reactor having a volume of 1 m³ or more. 5.The process according to claim 1, wherein enzymatic hydrolysis time is 5to 150 hours.
 6. The process according to claim 1, wherein the enzymecomposition used retains activity for 30 hours or more.
 7. The processaccording to claim 1, wherein the hydrolysis is conducted at atemperature of 45° C. or more.
 8. The process according to claim 1,wherein the enzyme composition is derived from a fungus, or the enzymecomposition comprises a fungal enzyme.
 9. The process according to claim1, wherein dry matter content in the enzymatic hydrolysis is 10 wt % ormore.
 10. The process according to claim 1, wherein the dry mattercontent in the enzymatic hydrolysis is 14 to 33% wt %.
 11. The processaccording to claim 1 wherein the enzymatic hydrolysis takes place in abatch, fed batch and/or continuous culture reactor.
 12. The processaccording to claim 1 wherein oxygen is introduced as air.
 13. Theprocess according to claim 8, wherein the fungus is of the genusRasamsonia, or the fungal enzyme is a Rasamsonia enzyme.
 14. The processaccording to claim 1, wherein the second part of time is 12% to 50% oftotal enzymatic hydrolysis time.
 15. The process according to claim 1,wherein during the first part of time dissolved oxygen in thelignocellulosic material is at a concentration that is at least 50% lessthan during the second part of time, and during the second part of timeoxygen is added to the lignocellulosic material continuously.
 16. Theprocess according to claim 1, wherein dissolved oxygen concentration inthe lignocellulosic material is maintained in the range of 0.02 mol/m³to 0.045 mol/m³, and wherein the dissolved oxygen is measured undernormal atmospheric pressure and at about 62° C.
 17. The processaccording to claim 13, wherein the fungus is Rasamsonia emersonii, orthe fungal enzyme is a Rasamsonia emersonii enzyme.
 18. The processaccording to claim 1, wherein no oxygen is added to the lignocellulosicmaterial during the first period of time.